Peptidyl compounds

ABSTRACT

Compounds of general formula (I): ##STR1## have utility as inhibitors of matrix metalloproteinases and TNF.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 08/539,578,filed Oct. 5, 1995 (Abn).

FIELD OF THE INVENTION

This invention relates to a novel class of peptidyl derivatives, toprocesses for their preparation, and to their use in medicine.

BACKGROUND TO THE INVENTION

In normal tissues, cellular connective tissue synthesis is offset byextracelluar matrix degradation, the two opposing effects existing indynamic equilibrium. Degradation of the matrix is brought about by theaction of proteinases released from resident connective tissue cells andinvading inflammatory cells, and is due, in part, to the activity of atleast three groups of metalloproteinases. These are the collagenases(interstitial collagenase, MMP-1; PMN collagenase, MMP-8, collagenase-3,MMP13), the gelatinases (gelatinase A, MMP-2, 72 kDa-gelatinase, Type IVcollagenase; gelatinase B, MMP-9, 92 kDa-gelatinase, Type IVcollagenase) and the stromelysins (proteoglycanase, MMP-3,stromelysin-1, transin; stromelysin-2, MMP:10; stromelysin-3, MMP:11).Normally these catabolic enzymes are tightly regulated at the level oftheir synthesis and secretion and also at the level of theirextracellular activity, the latter through the action of specificinhibitors, such as TIMP (tissue inhibitors of metalloproteinase), whichform inactive complexes with metalloproteinases, and more generalproteinase inhibitors such as a₂ -macroglobulins.

The accelerated, uncontrolled breakdown of connective tissues bymetalloproteinase catalysed resorption of the extracellular matrix is afeature of many pathological conditions such as rheumatoid arthritis,osteoarthritis, septic arthritis, corneal, epidermal or gastriculceration; tumour metastasis or invasion; periodontal disease,proteinuria, coronary thrombosis associated with atherosclerotic plaquerupture and bone disease. The inhibitors claimed herein may also beuseful in preventing the pathological squaelae following a traumaticinjury that could lead to a permanent disability. These compounds mayalso have utility as a means for birth control by preventing ovulationor implantation. It can be expected that the pathogenesis of suchdiseases is likely to be modified in a beneficial manner by theadministration of metalloproteinase inhibitors and numerous compoundshave been suggested for this purpose [for a general review see R C Wahl,et al Ann. Rep, Med. Chem. 25:175-184, Academic Press Inc., San Diego(1990)].

A number of small peptide like compounds which inhibitmetalloproteinases have been described. Perhaps the most notable ofthese are those relating to angiotensin converting enzyme (ACE) wheresuch agents act to block the conversion of the decapeptide angiotensin Ito angiotensin II, a potent pressor substance. Compounds of this typeare described in EP-A-0012401. Also, related mercaptoamide peptidylderivatives have shown ACE inhibitor activity in vitro and in vivo (H NWeller et al (1984), Biochem Biophys. Res. Comm., 125 (1):82-89).

TNFα is a cytokine which is produced initially as a cell-associated 28kD precursor. It is released as an active, 17 kD form (D-M Jue et al,(1990) Biochemistry, 29:8371-8377), which can mediate a large number ofdeleterious effects in vivo. When administered to animals or humans itcauses inflammation, fever, cardiovascular effects, haemorrhage,coagulation and acute phase responses, similar to those seen duringacute infections and shock states. Chronic administration can also causecachexia and anorexia. Accumulation of excessive TNFα can be lethal.There is considerable evidence from animal model studies that blockingthe effects of TNFα with specific antibodies can be beneficial in acuteinfections, shock states, graft versus host reactions and autoimmunedisease. TNFα is also an autocrine growth factor for some myelomas andlymphomas and can act to inhibit normal heamatopoiesis in patients withthese tumours.

Preventing the production or action of TNFα is, therefore, predicted tobe a potent therapeutic strategy for many inflammatory, infectious,immunological or malignant diseases. These include, but are notrestricted to, septic shock, haemodynamic shock and sepsis syndrome(Mathison et al (1988) J. Clin. Invest. 81:1925-1937; Miethke et al(1992), J. Exp. Med. 175:91-98), post-ischaemic reperfusion injury,malaria (Grau et al (1989), Immunol. Rev. 112:49-70); mycobacterialinfection (Barnes et al (1992) Infect. Imm. 60:1441-6), meningitis,psoriasis, congestive heart failure, fibrotic disease, cachexia, graftrejection, cancer, autoimmune disease, rheumatoid arthritis, multiplesclerosis, radiation damage, toxicity following administration ofimmunosuppressive monoclonal antibodies such as OKT3 or CAMPATH-1 andhyperoxic alveolar injury.

Current clinical anti-TNFα strategies involve the use of corticosteroidssuch as dexamethasone, and the use of cyclosporin-A or FK506, which arenon-specific inhibitors of cytokine gene transcription.Phosphodiesterase inhibitors such as pentoxyfilline have been shown tobe more specific inhibitors of TNFα gene transcription (Endres S. (1991)Immunol. 72:56-60, Schandene et al (1992), Immunol. 76:30-34, Alegre ML, et al (1991); Transplantation 52:674-679, Bianco et al (1991) Blood78:1205-1221). Thalidomide has also been shown to inhibit TNFαproduction by leucocytes (Sampajo et al (1991), J. Exp. Med.173:699-703). In experimental settings, anti-TNFα monoclonal antibodies,soluble TNF receptors and soluble TNF receptor/immunoadhesins have beenshown to specifically inhibit the effects of TNFα action (Bagby et al(1991) J. Infect. Dis. 163:83-88, Charpentier et al. (1991) Presse-med.20:2009-2011, Silva et al (1990) J. Infect. Dis. 162:421-427; Franks etal (1991) Infect. Immun. 59:2609-2614, Tracey et al (1987) Nature330:662-664; Fischer et al (1992) PNAS USA in press, Lesslauer et al(1991) Eur. J. Immunol. 21:2883-2886, Ashkenazi et al (1991) PNAS USA88:10535-10539).

It has recently been shown that the effects of TNF are mediated by twopeptides, TNFα and TNFβ. Although these peptides have only 30% homologywith each other, they activate the same receptors and are encoded byimmediately adjacent genes. As used herein, the term tumour necrosisfactor or TNF therefore means tumour necrosis factor α and peptideshaving a high degrees of sequence homology with, or substantiallysimilar physiological effects to, TNFα, for example TNFβ. One of theobjectives of the present invention is to provide compounds whichsubstantially inhibit the release of TNF from cells, and therefore maybe used in the treatment of conditions mediated by TNF. Such usesinclude, but are not limited to, the treatment of inflammation, fever,cardiovascular effects, haemorrhage, coagulation and acute phaseresponse, cachexia and anorexia, acute infections, shock states, graftversus host reactions and autoimmune disease.

Compounds which have the property of inhibiting the action ofmetalloproteinases involved in connective tissue breakdown such ascollagenase, stromelysin and gelatinase have been shown to inhibit therelease of TNF both in vitro and in vivo (A J H Gearing et al (1994),Nature, 370:555-557; G M McGeehan et al (1994), Nature, 370:558-561; M JCrimmin et al, WO 93/20047). All of these reported inhibitors contain ahydroxamic acid zinc binding group.

It is, therefore, a further object of this invention to providecompounds which, in addition to inhibiting TNF release, also inhibit theaction of MMPs, and hence may be used in the treatment of patients whosuffer from conditions mediated by TNF and/or MMPs.

As appreciated by those of skill in the art the significant proportionof homology between human fiberblast collagenase, stromelysin andgelatinase leads to the possibility that a compound that inhibits oneenzyme may to some degree inhibit all of them.

Compounds that inhibit collagenase, which possess structural portionsakin to those of the instant invention include those encompassed by U.S.Pat. No. 4,511,504 issued Apr. 16, 1985; U.S. Pat. No. 4,568,666, issuedFeb. 4, 1986.

Compounds of related structure that are claimed to inhibit stromelysin(proteoglycanase) are encompassed by U.S. Pat. No. 4,771,037, issuedSep. 13, 1988. The applicants believe that stromelysin and collagenaseinhibitors have utility in preventing articular cartilage damageassociated with septic arthritis. Bacterial infections of the joints canelicit an inflammatory response that may then be perpetuated beyond whatis needed for removal of the infective agent resulting in permanentdamage to structural components. Bacterial agents have been used inanimal models to elicit an arthritic response with the appearance ofproteolytic activities. See J. P. Case et al (1989), J. Clin. Invest.,84:1731-40; R. J. Williams et al (1990), Arth. Rheum., 33:533-41.

The applicants also believe that inhibitors of stromelysin, collagenaseand gelatinase will be useful to control tumour metastasis, optionallyin combination with current chemotherapy and/or radiation. See L. MMatrisian et al (1986), Proc. Natl. Acad. Sci., USA, 83:9413-7; S. M.Wilhelm et al (1987), Ibid. 84:6725-29; Z. Werb et al (1989), J. CellBiol., 109:872-889; L. A. Liotta et al (1983), Lab. Invest., 49:636-649;R. Reich et al in Metatasis; Ciba Foundation Symposium, Wiley,Chicester, 1988, pp. 193-210.

Secreted proteinases such as stromelysin, collagenase and gelatinaseplay an important role in processes involved in the movement of cellsduring metastasic tumour invasion. Indeed, there is also evidence thatthe matrix metalloproteinases are overexpressed in certain metastatictumour cell lines. In this context, the enzyme functions to penetrateunderlying basement membranes and allow the tumour cell to escape fromthe site of primary tumour formation and enter the circulation. Afteradhering to blood vessel walls, the tumour cells use these samemetalloproteinases to pierce underlying basement membranes and penetrateother tissues, thereby leading to tumour metastasis. Inhibition of thisprocess would prevent metastasis and improve the efficacy of currenttreatments with chemotherapeutics and/or radiation. These inhibitorsshould also be useful for controlling periodontal diseases, such asgingivitis. Both collagenase and stromelysin activities have beenisolated from fibroblasts derived from inflamed gingiva (V. J. Uitto etal (1981), J. Periodontal Res., 16:417-424). Enzyme levels have beencorrelated to the severity of gum disease; C. M. Overall et al (1987),J. Periodontal Res., 22:81-88.

Proteolytic processes have also been observed in the ulceration of thecornea following alkali burns (S. I. Brown et al (1969), Arch.Opthalmol., 81:370-373). Mercapto-containing peptides do inhibit thecollagenase isolated from alkali-burned rabbit cornea (F. R. burns et al(1989), Invest. Opthalmol, 30:1569-1575). Treatment of alkali-burnedeyes or eyes exhibiting corneal ulceration as a result of infection withinhibitors of these metalloendoproteinases in combination with sodiumcitrate or sodium ascorbate and/or antimicrobials may be effective inpreventing developing corneal degradation.

Stromelysin has been implaced in the degradation of structuralcomponents of the glomerular basement membrane (GBM) of the kidney, themajor function of which is to restrict passage of plasma proteins intothe urine (W. H. Baricos et al (1989), Biochem. J., 254:609-612).Proteinuria, a result of glomerular disease, is excess protein in theurine caused by increased permeability of the GBM to plasma proteins.The underlying causes of the increased GBM permeability are unknown, butproteinases including stromelysin may play an important role inglomerular diseases. Inhibition of this enzyme may alleviate theproteinura associated with kidney malfunction.

It is suggested that inhibition of stromelysin activity may prevent therupturing of atherosclerotic plaques leading to coronary thrombosis. Thetearing or rupture of atherosclerotic plaques is the most common eventinitiating coronary thrombosis. Destabilisation and degradation of theconnective tissue matrix surrounding these plaques by proteolyticenzymes or cytokines released by infiltrating inflammatory cells hasbeen proposed as a cause of plaque fissuring. Such tearing of theseplaques can cause an acute thrombolytic event as blood rapidly flows outof the blood vessel. High levels of stromelysin RNA message have beenfound to be localised to individual cells in atherosclerotic plaquesremoved from heart transplant patients at the time of surgery (A. M.Henney et al (1991), Proc. Nat'l. Acad. Sci. USA, 88:8154-8158).Inhibition of stromelysin by these compounds may aid in preventing ordelaying the degradation of the connective tissue matrix that stabilisesthe atherosclerotic plaques, thereby preventin events leading to acutecoronary thrombosis.

It is also believed that specific inhibitors of stromelysin andcollagenase should be useful as birth control agents. There is evidencethat expression of metalloproteinases, including stromelysin andcollagenase, is observed in unfertilised eggs and zygotes and at furthercleavage stages and increased at the blastocyst stage of fetaldevelopment and with endoderm differentiation (C. A. Brenner et al(1989), Genes & Develop., 3:848-59). By analogy to tumour invasion, ablastocyst may express metalloproteinases in order to penetrate theextracelluar matrix of the uterine wall during implantation. Inhibitionof stromelysin and collagenase during these early development processesshould presumably prevent normal embryonic development and/orimplantation in the uterus. Such intervention would constitute a novelmethod of birth control. In addition there is evidence that collagenaseis important in ovulation processes. In this example, a covering ofcollagen over the apical region of the follicle must be penetrated inorder for the ovum to escape. Collagenase has been detected during thisprocess and an inhibitor has been shown to be effective in preventingovulation (J. F. Woessner et al (1989), Steroids, 54:491-499). There mayalso be a role for stromelysin activity during ovulation (C. K. L. Tooet al (1984), Endocrin., 115:1043-1050).

Collagenolytic and stromelysin activity have also been observed indystrophic epidermolysis bullosa (A. Kronberger et al (1982), J. Invest.Dermatol., 79:208-211; D. Sawamura et al (1991), Biochem. Biophys. Res.Commun., 184:1003-8). Inhibition of metalloendoproteinases should limitthe rapid destruction of connective components of the skin.

In addition to extracelluar matrix comprising structural components,stromelysin can degrade other in vivo substrates including theinhibitors a₁ -proteinase inhibitor and may therefore influence theactivities of other proteinases such as elastase (P. G. Winyard et al(1991), FEBS Letts., 279, 1:92-94). Inhibition of the matrixmetalloendoproteinases may potentiate the antiproteinase activity ofthese endogenous inhibitors.

From recent publications it is evident that several new enzymes of theMMP family have been identified, some of which maybe important indisease. Collagenase 3, an enzyme unique to breast carcinoma cells mayhave utility in breast cancer (J M P Freije et al (1994), J. Biol.Chem., 269 (24): 16766-16773), whilst MT-MMP, another member of the MMPfamily has been shown to be a key enzyme in the activaton of gelatinaseA (H Sato et al (1994), Nature, 370:61-65). Gelatinase A is an importantenzyme in the growth and metastasis of tumours (such as defined above).

The degradation of b-Amyloid Precusor Protein (APP) has been shown togenerate amyloid plaques, a major constitutent of the senile plaques,found in patients with Alzheimers Disease (AD). Two recent publicationshave identified metalloproteinase enzymes that cleave APP to the amyloidplaque (CR Abraham et al (1994), Biochemistry, 33:192-199; G Huber et al(1994), Biochem. Biophys. Res. Comm., 201 (1):45-53).

As appreciated by those of skill in the art, the significant proportionof homology between these new enzymes and other MMPs leads to thepossibility that a compound that inhibits one enzyme may to some degreeinhibit these new enzymes. Therefore, inhibitors encompassed in thisinvention may be useful in the diseases in which these new enzymes areimplicated.

SUMMARY OF THE INVENTION

The invention encompasses novel mercaptoalkylpeptidyl compounds offormula (I) which are useful inhibitors of matrix metalloproteinasesand/or TNFα mediated diseases including degenerative diseases (such asdefined above) and certain cancers.

In a first aspect of the invention there is provided a compound ofgeneral formula (I): ##STR2## Wherein:

R¹ is C₁₋₆ alkyl, C₂₋₆ alkenyl, --C₁₋₆ alkyl-aryl, aryl, --C₁₋₆alkyl-heteroaryl, heteroaryl or --C₁₋₆ alkyl-AR⁹ where A represents O,NR⁹ or S(O)_(m) where m=0-2, and R⁹ is H, C₁₋₄ alkyl, aryl, heteroaryl,--C₁₋₄ alkyl-aryl or --C₁₋₄ alkyl-heteroaryl; if A═NR⁹ the groups R⁹ maybe the same or different;

R² is H or C₁₋₆ alkyl;

R³ is [Alk]_(n) R⁶ where Alk is C₁₋₆ alkyl or C₂₋₆ alkenyl and n is zeoor 1;

X is NR⁴ R⁵ where either R⁴ is hydrogen or C₁₋₆ alkyl optionallysubstituted by amino (NH₂), aryl, arylamino, protected amino, di(C₁₋₆alkyl)amino, mono(C₁₋₆ alkyl)amino, CO₂ H, protected carboxyl,carbamoyl, mono(C₁₋₆ alkyl)carbamoyl or di(C₁₋₆ alkyl)carbamoyl, and R⁵is hydrogen or C₁₋₆ alkyl; or NR⁴ R⁵ forms a ring such as pyrrolidino,piperidino or morpholino;

R⁷ is hydrogen or R¹⁰ CO where R¹⁰ is C₁₋₄ alkyl, --C₁₋₄ alkyl-aryl,--C₁₋₄ alkyl-heteroaryl, cyclo(C₃₋₆)alkyl, --C₁₋₄alkyl-cyclo(C₃₋₆)alkyl, C₂₋₆ alkenyl, --C₂₋₆ alkenyl-aryl, aryl orheteroaryl;

R⁸ is aryl (substituted with R¹¹), heteroaryl (optionally substitutedwith R¹¹), C₁₋₄ alkyl-R¹¹, --C₁₋₄ alkyl-aryl (substituted with R¹¹),--C₁₋₄ alkyl-heteroaryl (optionally substituted with R¹¹),cyclo(C₃₋₆)alkyl (optionally substituted with R¹¹), cyclo(C₃₋₆)alkenyl(optionally substituted with R¹¹), --C₁₋₄ alkyl-cyclo(C₃₋₆)alkyl(optionally substituted with R¹¹), or any of the three groups ##STR3##where p is 1 or 2 and B and C are independently selected for O, S,C(R⁹)₂ and NR⁹ ;

R⁶ is AR⁹, cyclo(C₃₋₆)alkyl, cyclo(C₃₋₆)alkenyl, C₁₋₆ alkyl, --C₁₋₆alkoxy-aryl, benzyloxyaryl, aryl, heteroaryl, --C₁₋₃ alkyl-heteroaryl,--C₁₋₃ alkyl-aryl, --C₁₋₆ alkyl-COOR⁹, --C₁₋₆ alkyl-NHR, CONHR, NHCO₂ R,NHSO₂ R or NHCOR, R being defined as for R¹⁰ ;

R¹¹ is SO₂ R¹³, SR⁷, SR⁹, COR¹³, N(R⁹)₂, NR⁹ R¹², OR⁹, succinimido orphthalimido;

R¹² is H or COR⁹, CO₂ R⁹ (where R⁹ is not H), CONHR⁹ or SO₂ R⁹ (where R⁹is not H); and

R¹³ is OH, OC₁₋₄ alkyl, O-C₁₋₄ alkyl-aryl, N(R⁹)₂ (in which the R⁹ s arethe same or different), C₁₋₄ alkyl, aryl, heteroaryl, --C₁₋₄ alkyl-arylor --C₁₋₄ alkyl-heteroaryl;

the compound being in the form of a non-salt, salt, solvate or hydrate.Preferred compounds of the invention include those in which,independently or in any combination have:

R¹ is C₁₋₆ alkyl or C₁₋₄ alkylAR⁹ where A is S(O)_(m), NR⁹, or O andm=0,1 or 2, and

R⁹ is H, C₁₋₄ alkyl or aryl;

R² is H or C₁₋₄ alkyl;

R³ is [Alk]_(n) R⁶ where n=0 or 1, Alk is C₁₋₄ alkyl and R⁶ is C₁₋₄alkyl, C₁₋₃ alkylaryl, C₁₋₃ alkylheteroaryl or AR⁹ ;

R⁴ is H;

R⁵ is H or C₁₋₆ alkyl;

NR⁴ R⁵ may form a 5-7 membered ring such as a pyrrolidine, piperidine ormorpholine;

R⁷ is H or R¹⁰ CO where R¹⁰ is C₁₋₄ alkyl;

R⁸ is C₁₋₄ alkylR¹¹, C₁₋₄ alkenylR¹¹, Cyclo(C₃₋₆)alkylR¹¹ ;

R¹¹ is COR¹³, NR⁹ R¹², N(R⁹)₂, succinimido or phthalimido,

R¹² is COR⁹, CO2R⁹ (provided R9 is not H), or SO₂ R⁹ (provided R9 is notH); and R¹³ is OH, OC₁₋₄ alkyl or N(R⁹)₂ ;

Compounds of the invention have IC₅₀ values below 50 mM against the MMPenzymes and/or below 50 mM in the whole cell assay of TNF inhibition. Itwill be appreciated that the compounds according to the invention cancontain one or more asymmetrically substituted carbon atoms, for examplethose marked with an asterisk in formula (1). The presence of one ormore of these asymmetric centres in a compound of formula (1) can giverise to stereoisomers, and in each case the invention is to beunderstood to extend to all such stereoisomers, including enantiomersand diastereomers, and mixtures including racemic mixtures thereof. Inthe formulae herein, the ˜ line is used at a potential asymmetric centreto represent the possibility of R- and S- configurations, the < line andthe . . . line to represent a unique configuration at an asymmetriccentre.

As used in this specification, alone or in combination, the term "C₁₋₆alkyl" refers to a straight or branched chain alkyl moiety having fromone to six carbon atoms, including for example, methyl, ethyl, propyl,isopropyl, butyl, t-butyl, pentyl, hexyl and the like.

The term "C₁₋₄ alkyl" refers to a straight or branched chain alkylmoiety having from one to four carbon atoms, including for example,methyl ethyl, propyl, isopropyl, butyl, t-butyl and the like.

The term "C₂₋₆ alkenyl" refers to a straight or branched chain alkylmoiety having two to six carbon atoms and having in addition one doublebond, of either E or Z stereochemistry where applicable. This term wouldinclude for example, vinyl, 1-propenyl, 1- and 2-butenyl,2-methyl-2-propenyl etc.

The term "cyclo(C₃₋₆)alkyl" refers to a saturated alicyclic moietyhaving from three to six carbon atoms and includes for examplecyclopropyl, cyclobutyl, cyclopentyl, cyclohexy and the like.

The term "cyclo(C₃₋₆) alkenyl" refers to an alicyclic moiety having fromthree to six carbon atoms and having in addition one double bond. Thisterm would include for example cyclopentenenyl or cyclohexenyl.

The term "aryl" means an optionally substituted phenyl or naphthyl groupwith the substituent(s) being selected, for example, from halogen,trifluoromethyl, C₁₋₆ alkyl, alkoxy, phenyl and the like. The term"halogen" means fluorine, chlorine, bromine or iodine.

The terms "protected amino" and "protected carboxy" mean amino andcarboxy groups which are protected in a manner familiar to those skilledin the art. For example, an amino group can be protected by abenzyloxycarbonyl, tert-butoxycarbonyl, acetyl or like groups, or in theform of a phthalimido or like group. A carboxyl group can be protectedin the form of a readily cleavable ester such as the methyl, ethyl,benzyl or tert-butyl ester.

The term "alkoxy" refers to a straight chain or branched chain alkoxygroup containing a maximum of six carbon atoms, such as methoxy, ethoxy,propoxy, isopropoxy, butoxy, tert-butoxy and the like.

The term "C₀₋₄ alkyl" refers to a bond or straight or branched chainalkyl moiety having from up to four carbon atoms, including for example,methyl, ethyl, propyl, isopropyl and the like.

The term "heteroaryl" refers to aromatic ring systems of five to tenatoms of which at least one atom is selected from the group, O, N or S.

Salts of compounds of formula (I) include pharmaceutically acceptablesalts, for example acid addition salts derived from inorganic or organicacids, such as hydrochlorides, hydrobromides, p-toluenesulphonates,phosphates, sulphates, perchlorates, acetates, trifluoroacetates,propionates, citrates, malonates, succinates, lactates, oxalates,tartrates, and benzoates.

Salts may also be formed with bases. Such salts include salts derivedfrom inorganic or organic bases, for example alkali metal salts such asmagnesium or calcium salts, and organic amine salts such as morpholine,piperidine, dimethylamine, or dimethylamine salts.

When the "protected carboxy" group in compound of the invention is anesterified carbozyl group, it may be a metabolically labile ester offormula CO₂ R¹⁴ where R¹⁴ may be an ethyl, benzyl, phenethyl,phenylpropyl, α- or β-naphthyl, 2,4-dimethylphenyl, 4-tert-butylphenyl,2,2,2-trifluoroethyl, 1-(benzyloxy)benzyl, 1-(benxyloxy)ethyl,2-methyl-1-propionyloxypropyl, 2,4,6-trimethylbenzyloxymethyl orpivaloyloxymethyl group.

Compounds of the general formula (I) may be prepared by any suitablemethod known in the art and/or by the following processes, which itselfforms part of the invention.

According to a second aspect of the invention, there is provided aprocess for preparing a compound of general formula (I) as definedabove. It will be appreciated that where a particular stereoisomer offormula (I) is required, the synthetic processes described herein may beused with the appropriate homochiral starting material and/or isomersmay be resolved from mixtures using conventional separation techniques(e.g., HPLC).

The compounds according to the invention may be prepared by thefollowing process. In the description and formulae below, the groups R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, A, B, C, and Xare as defined above, except where otherwise indicated. It will beappreciated that functional groups, such as amino, hydroxyl, or carboxylgroups, present in the various compounds described below, and which itis desired to retain, may need to be in protected form before anyreaction is initiated. In such instances, removal of the protectinggroup may be the final step in a particular reaction. Suitableprotecting groups for such functionality apparent to those skilled inthe art. For specific details see "Protective Groups in OrganicSynthesis", Wiley Interscience, T W Greene, PGM Wuts. Thus a process forpreparing compounds of general formula (I) comprises the steps of:

deprotecting (for example by hydrolysis) a compound of general formula(II) ##STR4## wherein R⁷ represents a suitable protecting group (eg tertbutyl or acetate). It will be appreciated that where a particularstereoisomer of formula (I) is required, this may be obtained byconventional resolution techniques such as high performance liquidchromatography. Where desired, however, appropriate homochiral startingmaterials may be used in the coupling reaction to yield a particularstereoisomer of formula (I). This is exemplified below.

Intermediates of general formula (II) may be prepared by coupling anacid of formula (III) ##STR5## wherein R⁷ and R⁸ are as defined above,or an active derivative thereof, with an amine of formula (IV) ##STR6##Active derivatives of acids of formula (III) include for example acidanhydrides or acid halides, such as acid chlorides.

The coupling reaction may be performed using standard conditions foramination reactions of this type. Thus, the reaction may be achieved ina solvent, for example an inert organic solvent such as an ether, eg. acyclic ether such as tetrahydrofuran, an amide eg. a substituted amidesuch as dimethyformamide, or a halogenated hydrocarbon such asdichlormethane at a low temperature eg. -30° C. to ambient temperature,such as -20° C. to 0° C., optionally in the presence of as base, eg. anorganic base such as an amine, eg. triethylamine or a cyclic amine suchas N-methylmorpholine. Where an acid of formula (III) is used, thereaction may additionally be performed in the presence of a condensingagent, for example a diimide such as N,N'-dicyclohexylcarbodiimide,advantageously in the presence of a triazole such as1-hydroxybenzotriazole. Alternatively, the acid may be reacted with achloroformate for example ethylchloroformate, prior to reaction with theamine of formula (IV).

The amine of general formula (IV) may be prepared by coupling an acid offormula (V), or an active derivative thereof ##STR7## with an amine offormula (VI) ##STR8## followed by removal of any protecting groups.

Active derivates of acids for formula (V) include for example acidanhydrides or acid halides such as acid chlorides as outlined earlier.

Amino acids and their dervatives as depicted by general formulae (V) and(VI) can be obtained in chiral or racemic form. In the chiral form theyprovide asymmetric building blocks for the chiral synthesis of compoundsof general formula (1). Many of these derivatives can be readilyobtained from commercially available starting materials using methodsknown to those skilled in the art. (See "The Practice of PeptideSynthesis" by M. Bodanszk et al, Springer Verlag, New York, 1984, P. L.Durette, WO92/21360).

As a further extension to the present invention compounds of generalformula (II) may be prepared by nucleophilic substitution of compoundsof general formula (VII) ##STR9## wherein R¹⁵ represents a suitableleaving group (e.g. a halogen such as bromide, or an alkylsulphonateester such as methansulphonate) with a thiol of general formula (VIII)

    R.sup.7 SH                                                 (VIII)

Wherein R⁷ represents a suitable protecting group (eg. tert-butyl oracetate), using standard conditions known to those skilled in the art asexemplified in WO 90/05719. Thiols of general formula (VIII) may beobtained from commercially available starting materials using methodsknown to those skilled in the art. Many thiols of general formula (VIII)are also commercially available.

Compounds of general formula (VII) may be prepared by coupling an acidof general formula (IX) ##STR10## wherein R¹⁵ and R⁸ are as definedabove (or suitably protected versions thereof) or an active derivativethereof, with an amine of formula (IV) using similar coupling conditionsto those described for the preparation of compounds of formula (II).Carboxylic acids of the structure depicted in formulae (III) and (IX)can be obtained in chiral or racemic form. Many of these derivatives canbe readily obtained from commercially available starting materials usingmethods known to those skilled in the art (see WO 90/05719).

As a further extension to the present invention, intermediates ofgeneral formula (II) may be prepared by coupling an acid of formula (X)##STR11## wherein R¹, R⁷ and R⁸ are as defined above, or an activederivative thereof, with an amine of formula (VI) by the proceduredescribed previously.

Acids of general formula (X) may in turn be preapred by coupling an acidof formula (III), or an active derivative thereof with an amine offormula (VI), where X═OH or a suitably protected derivative theroffollowed by removal of any protecting groups.

Active derivates of acids for formula (V) include for example acidanhydrides or acid halides such as acid chlorides as outlined earlier.

Compounds of formula (I) may also be prepared by interconversion ofother compounds of formula (I). Thus, for example, a compound of formula(I) wherein R¹ is a C₁₋₆ alkyl group may be prepared by hydrogenation(using palladium on carbon in suitable solvent, such as an alcohol--egethanol) of a compound of formula (I) wherein R¹ is a C₂₋₆ alkenylgroup. A further example would include a compound of formula (I) whereinR⁷ is a group R¹⁰ CO may be prepared by acylation (using a suitable acidchloride R¹⁰ COCl, in the presence of a base such as a triethylamine ina suitable solvent, such as a chlorinated solvent--eg dichloromethane)of a compound of formula (I) wherein R⁷ is H.

Any mixtures of final products or intermediates obtained can beseparated on the basis of the pysico-chemical differences of theconstituents, in known manner, into the pure final products orintermediates, for example by chromatography, distillation, fractionalcrystallization, or by formation of a salt if appropriate or possibleunder the circumstances.

The compounds according to the invention exhibit in vitro inhibitingactivities with respect to stromelysin, collagenase and gelatinase.Compounds according to the invention also exhibit in vitro inhibition ofTNFα release. The activity and selectivity of the compounds may bedetermined by use of the appropriate enzyme inhibition test, for exampleas described in Example A hereinafter.

This invention also relates to a method of treatment for patients(including man and/or mammalian animals raised in the dairy, meat or furindustries or as pets) suffering from disorders or diseases which can beattributed to matrix metalloproteinases and/or TNFα as previouslydescribed, and more specifically, a method of treatment involving theadministration of the matrix metalloproteinase inhibitors of formula (I)as the active constituents.

Accordingly, the compounds of formula (I) can be used among other thingsin the treatment of osteoarthritis and rheumatoid arthritis, and indiseases and indications resulting from the over-expression of thesematrix metalloproteinases such as found in certain metastatic tumourcell lines.

As mentioned above, compounds of formula (I) are useful in human orveterinary medicine since they are active as inhibitors of TNFα andMMPs. Accordingly in another aspect, this invention concerns:

a method of management (by which is meant treatment or prophylaxis) ofdisease or conditions mediated by TNFα and/or MMPs in mammals, inparticular in humans, which method comprises administering to the mammalan effective amount of a compound of formula (I) above, or apharmaecutically acceptable salt thereof; and a compound of formula (I)for use in human or veterinary medicine, particularly in the management(by which is meant treatment or prophylaxis) of diseases or conditionsmediated by TNFα and/or MMPs; and

the use of a compound of formula (I) in the preparation of an agent forthe management (by which is meant treatment or prophylaxis) of diseasesor conditions mediated by TNFα and/or MMPs.

The disease or conditions referred to above include inflammation, fever,cardiovascular effects, haemorrhage, coagulation and acute phaseresponse, cachexia and anorexia, acute infections, shock states, graftversus host reactions and autoimmune disease; and those involving tissuebreakdown such as bone resportion, inflammatory diseases, dermatologicalconditions, tumour growth, angiogenesis and invasion by secondarymetastases, in particular rheumatoid arthritis, osteoarthritis,periodontitis, gingivitis, corneal ulceration, tumour growth,angiogenesis and invasion by secondary metastases.

For the treatment of rheumatoid arthritis, osteoarthritis, and indiseases and indications resulting from the over-expression of matrixmetalloendoproteinases such as found in certain metastatic tumour celllines or other diseases mediated by the matrix metalloendoproteinases orincreased TNFα production, the compounds of formula (I) may beadministered orally, topically, parenterally, by inhalation spray orrectally in dosage unit formulations containing non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles. The termparenteral as used herein includes subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques. Inaddition to the treatment of warm-blooded animals such as mice, rats,horses, cattle, sheep, dogs, cats etc, the compounds of the inventionare effective in the treatment of humans.

The pharmaceutical composition containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspension, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavouring agents, colouring agents andpreserving agents in order to provide pharmaecutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example corn starch, or alginic acid; bindingagents, for example starch, gelatin or acacia, and lubricating agents,for example magnesium stearate, stearic acid or talc. The tablets may beuncoated or they may be coated by known techniques to delaydisintegration and absorption in the gastointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyeryl distearate maybe employed. They may also be coated by the techniques described in theU.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osomotictherapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsuleswhere in the active ingredient is mixed with an inert solid diluent, forexample calcium carbonate, calcium phosphate or kaolin, or as solftgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methycellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally occuring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such a polyoxyethylene with partial esters derived from fattyacids and hexitol anhydrides, for example polyoxyethylene sorbitanmonooleate. The aqueous suspension may also contain one or morepreservatives, for example ethyl, or n-propyl, p-hyroxybenzoate, one ormore colouring agents, one or more flavouring agents, and one or moresweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavouring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified, for example sweetening, flavouringand colouring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occuring gums, for example gum acacia or gum tragancanth,naturally-occuring phosphatides, for example soya bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate and condensation products ofthe said partial esters with ethylene oxide, for example polyoxyethylenesorbitan monooleate. The emulsions may also contain sweetening andflavouring agents.

Syrups and elixirs may be formulated with sweetening agents, for examplegycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative and flavouring and colouringagents. The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be in a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butane diol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The compounds of formula (I) may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials are cocoa butter and polyethylene glycols.

For topical use, creams, ointments, jellies, solutions or suspensions,etc containing the compounds of Formula (I) are employed. (For purposesof this application, topical application shall include mouth washes andgargles.)

Dosage levels of the order of from about 0.05 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 2.5 mg to about 7 gms per patient perday). For example, inflammation may be effectively treated by theadministration of from about 0.01 to 50 mg of the compound per kilogramof body weight per day (about 0.5 mg to about 3.5 gms per patient perday).

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, aformulation intended for the oral administration of humans may vary fromabout 5 to about 95 percent of the total composition. Dosage unit formswill generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

The following Examples 1 to 79 illustrate the invention and theirpreparation (via the green Intermediates, as appropriate). Examples A toG illustrate test procedures.

In the Examples, the following abbreviations are used:

RT Room temperature

DCC Dicyclohexylcarbodiimide

EDC 1-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride

TNF.sub.α Tumour necrosis factor α

PMA Phorbol-13-myristate-12-acetate

ELISA Enzyme linked immunosorbent assay

INTERMEDIATE 1 (RS)-2-Bromo-4-methoxycarbonylbutanoic Acid

A solution of d-methyl-D,L-glutamic acid (6.0 g) (preparation accordingto Hanby et al, J. Chem. Soc. (1950), 51:3239) and potassium bromide(15.5 g) in aqueous sulfuric acid (1.25 M, 100 ml) was treated at 0° C.portionwise with sodium nitrite (4.0 g) over 1 h. The solution wasallowed to warm to RT and was stirred over 2 h, then extracted withethyl acetate (2×100 ml). The combined extracts were dried (MgSO₄) andevaporated in vacuo to give the title compound as a colourless oil (4.5g).

TLC R_(f) 0.14 (25% EtOAc-hexanes)

Similarly prepared were:

INTERMEDIATE 2 (RS)-2-Bromo-5-methoxycarbonylpentanoic Acid

From (RS)-2-amino-5-methoxycarbonylpentanoic acid (1.8 g) (preparationbased on the esterification procedure of Hanby et al, J. Chem. Soc.(1950), 51:3239) as a pale brown oil (1.95 g).

TLC R_(f) 0.30 (5% MeOH-CH₂ Cl₂)

INTERMEDIATE 3 (RS)-2-Bromo-6-methoxycarbonylhexanoic Acid

From (RS)-2-amino-6-methoxycarbonylhexanoic acid (3.93 g) (preparationbased on the esterification procedure of Hanby et al, J. Chem. Soc.(1950), 51:3239) as a colourless oil (4.39 g).

TLC R_(f) 0.26 (5% MeOH-CH₂ Cl₂)

INTERMEDIATE 4 (RS)-2-Acetylmercapto-3-methoxycarbonylpropionic Acid

A solution of potassium thiolacetate (1.48 g) in methanol (2 ml) wasadded to a solution of mono-methylmaleate (1.64 g) (prepared accordingthe procedure exemplified in J. Am. Chem. Soc. (1986), 108:3) and themixture was stirred overnight at RT. The solvent was removed byevaporation and the residue was partitioned between water (30 ml) anddichloromethane (30 ml), and the aqueous layer was then acidified to pH3with 2N aqueous hydrochloric acid. The layers were separated and theaqueous layer was extracted with dichloromethane (2×30 ml). The combinedorganic extracts were dried (MgSO₄) and evaporated in vacuo to give abrown oil. Purification by flash column chromatography (eluting with 10%methanol in dichloromethane) gave the title compound as a colourless oil(0.48 g).

TLC R_(f) 0.30 (10% MeOH-CH₂ Cl₂)

INTERMEDIATE 5 (RS)-2-Acetylmercapto-4-methoxycarbonylbutanoic Acid

Potassium thiolacelate (2.0 g) was added to a solution of intermediate 1(3.0 g) in ethanol (30 ml) and the mixture was stirred at RT overnight.The solution was evaporated in vacuo and the residue was partitionedbetween ethyl acetate (30 ml) and water (30 ml). The organic layer wasthen washed with saturated brine (30 ml), dried (MgSO₄) and evaporatedin vacuo to give the title compound as a colourless oil (1.83 g).

TLC R_(f) 0.46 (EtOAc)

Similarly prepared were:

INTERMEDIATE 6 (RS)-2-Acetylmercapto-5-methoxycarbonylpentanoic Acid

From Intermediate 2 (1.89 g) as a yellow oil (0.87 g).

TLC R_(f) 0.30 (5% MeOH-CH₂ Cl₂)

INTERMEDIATE 7 (RS)-2-Acetylmercapto-6-methoxycarbonylhexanoic Acid

From Intermediate 3 (4.3 g) as a yellow oil (2.78 g).

TLC R_(f) 0.23 (5% MeOH-CH₂ Cl₂)

INTERMEDIATE 8 (RS)-(1,1-Dimethylethyl) 2,4-dibromobutyrate

Bromine (31.3 ml, 0.54 mol) was added dropwise over 4h at 100° C. toneat stirring 4-bromobutyryl chloride (100 g, 0.54 mol). The resultingacid chloride was cooled then added dropwise at 0° C. to a stirredsolution of tert-butanol (240 ml) and triethylamine (64 ml, 0,461 mol)in anhydrous dichloromethane (600 ml). 2M Hydrochloric acid was addedand the layers separated. The organic portion was then washedsequentially with 10% sodium metabisulphite solution (2×500 ml), water(500 ml) and brine (500 ml), dried (MgSO₄) and evaporated in vacuo toprovide the title compound (120 g, 86%) as a brown liquid.

¹ H NMR (250 MHz; CDCl₃), Ref., TMS) 1.50 (9H, s), 2.45 (2H, q), 3.55(2H, t) and 4.40 (1H, dd).

Similarly prepared were:

INTERMEDIATE 9 (RS)-(1,1-Dimethylethyl) 2,5-dibromopentanoate

From 5-bromovaleryl chloride (75 g, 0.37 mol), as a brown oil (81 g,68%).

¹ H NMR (250 MHz; CDCl₃), Ref., TMS) 1.50 (9H, s), 1.8-2.3 (4H,m), 3.45(2H, t) and 4.18 (1H, dd).

INTERMEDIATE 10 (RS)-(1,1-Dimethylethyl) 2,6-dibromohexanoate

From 6-bromohexanoyl chloride (100 g, 0.47 mol), as a brown oil (133 g,87%).

¹ H NMR (250 MHz; CDCl₃, Ref., TMS) 1.50 (9H, s), 1.6-2.1 (6H, m), 3.4(2H, t) and 4.1 (1H, dd).

INTERMEDIATE 11 (RS)-(1,1-Dimethylethyl)2,4-bis-(acetylmercapto)butyrate

Potassium thiolacetate (1.51 g, 13.2 mmol) was added to a stirredsolution of intermediate 8 (2 g, 6.6 mmol) in methanol (25 ml) and themixture stirred at RT overnight. The mixture was diluted withdichloromethane (100 ml), washed with brine (2×50 ml), dried (MgSO₄) andevaporated in vacuo to a yellow oil. Purification by flash columnchromatography (eluting with 30% dichloromethane in hexane) provided thetitle compound (1.1 g, 57%) as a colourless oil.

TLC R_(f) 0.57 (CH₂ Cl₂)

Similarly prepared were:

INTERMEDIATE 12 (RS)-(1,1-Dimethylethyl)2,5-bis-(acetylmercapto)pentanoate

From intermediate 9 (2 g, 6.32 mmol), as a colourless oil (1.12 g, 57%)

TLC R_(f) 0.57 (CH₂ Cl₂)

INTERMEDIATE 13 (RS)-(1,1-Dimethylethyl)2,6-bis-(acetylmercapto)hexanoate

From intermediate 10 (2 g, 6.32 mmol), as a colourless oil (1.09 g, 57%)

TLC R_(f) 0.57 (CH₂ Cl₂)

INTERMEDIATE 14 (RS)-2,3-Bis-(acetylmercapto)propionic Acid

A solution of thiolacetic acid (1.12 g) in 1N aqueous potassium hyroxide(14.7 ml) was added dropwise to a solution of 2,3-dibromopropionic acid(1.71 g) in 1N aqueous potassium hydroxide (7.35 ml) and the mixture wasstirred 5 h at RT. The pH of the mixture was adjusted to 8-9 by theaddition of further 1N aqueous potassium hydroxide and the mixture wasstirred a further 2 h, then acidified to pH1-2 by the addition ofconcentrated hydrochloric acid and extracted with ethyl acetate (2×25ml). The combined extracts were dried (Na₂ SO₄) and evaporated to give ayellow oil. The product from two reactions was combined and purificationby flash column chromatography (eluting with 4% acetic acid-toluene)gave the title compound as a colourless oil (0.422 g).

TLC R_(f) 0.15 (5% AcOH-toluene)

INTERMEDIATE 15 (RS)-2,4-Bis-(acetylmercapto)butyric Acid

A solution of intermediate 11 (1.1 g, 3.7 mmol) in dichloromethane (50ml) was treated with trifluoroacetic acid (2.9 ml, 37 mmol) and themixture stirred at RT overnight. Water (50 ml) was added and the mixtureextracted with dichloromethane (3×40 ml). The combined organic extractswere then washed with water (50 ml) and brine (50 ml), dried (MgSO₄) andevaporated in vacuo to provide the product (870 mg, 98%) as a paleyellow oil.

TLC R_(f) 0.12 (25% MeOH-CH₂ Cl₂)

Similarly prepared were:

INTERMEDIATE 16 (RS)-2,5-Bis-(acetylmercapto)pentanoic Acid

From intermediate 12 (1.1 g 3.6 mmol), as a pale yellow oil (906 mg,100%)

TLC R_(f) 0.12 (25% MeOH-CH₂ Cl₂)

INTERMEDIATE 17 (RS)-2,6-Bis-(acetylmercapto)hexanoic Acid

From intermediate 13 (1.1 g, 3.6 mmol), as a pale yellow oil (895 mg,98%)

TLC R_(f) 0.12 (25% MeOH-CH₂ Cl₂)

INTERMEDIATE 18 4-Phthalimidobutanoic Acid

N-Carboethoxyphthalimide (10.96 g) was added on one portion to avigorously stirred solution of 4-aminobutanoic acid (5.16 g) and sodiumcarbonate (5.35 g) in water (150 ml) at RT. The mixture was stirreduntil essentially all the solid material had dissolved (30 min), then itwas filtered. The filtrate was acidified to pH1 with 6N aqueoushydrochloric acid (ca. 22 ml) and the white precipitate was collected byfiltration and washed thoroughly with water (150 ml). The solid wasdried in air, then in vacuo to give the title compound as a colourlesssolid (7.35 g).

¹ H NMR (250 MHz; CDCl₃, Ref., TMS) d 2.03 (2H. pent.) 2.42 (2H, t) 3.78(2H, t), 7.65-7.77 (2H, m), 7.81-7.90 (2H, m)

Similarly prepared were:

INTERMEDIATE 19

5-Phthalimidopentanoic acid

From 5-aminopentanoic acid (5.0 g) as a colourless solid (6.8 g).

¹ H NMR (250 MHz; CDCl₃, Ref., TMS) d 1.6-1.8 (4H, m) 2.20 (2H, t) 3.85(2H, t), 7.70-7.75 (2H, m), 7.85-7.95 (2H, m), 10.2 (1H, br s)

INTERMEDIATE 20

6-Phthalimidohexonoic acid

From 6-aminohexanoic acid (5.0 g) as a colourless solid (5.8 g).

¹ H NMR (60 MHz; CDCl₃, Ref., TMS) 1.5-2.4 (6H, m) 2.3 (2H, t) 3.80 (2H,t), 7.8-8.1 (4H, m), 10.4 (1H, br s)

INTERMEDIATE 21

2-(3-Phthalimidophenyl)acetic acid

From 2-(3-aminophenyl)acetic acid (3.0 g) as an off-white solid (4.0 g,72%).

TLC R_(f) 0.36 (7.5% MeOH-0.5% AcOH--CH₂ Cl₂)

INTERMEDIATE 22

Cis-3-Aminocyclopent-4,5-enecarboxylic acid

A solution of racemic lactam (50 g, 0.458 mol) in 2N hydrochloric acid(1000 ml) was heated under reflux for 1 h. The mixture was evaporated invacuo and the residue crystallised from acetone to provide thehydrochloride salt of the title compound (73 g, 97%) as a white solid.

This hydrochloride salt (20 g, 0.122 mol) was dissolved in water (300ml) and the stirred solution treated with amberlite (IRA-67) ionexchange resin until pH 7 was reached. The resin was then removed byfiltration, the solvent removed in vacuo and the residue crystallisedfrom acetone to provide the title compound (13.7 g, 88%) as a whitesolid.

INTERMEDIATE 23

CIS-3-Phthalimidocyclopent-4,5-enecarboxylic acid

From intermediate 22 hydrochloride (22.3 g, 0.136 mol), as a white solid(13.7 g, 39%).

TLC R_(f) 0.37 (1% AcOH-5% MeOH--CH₂ Cl₂)

INTERMEDIATE 24

Trans-Methyl 3-Phthalimidocyclopent-4,5-enecarboxylate

Intermediate 23 (23.2 g, 0.183 mol) and phthalic anhydride (27.03 g,0.183 mol) were powdered together and melted at 190° C. under nitrogenwith stirring. The mixture was allowed to cool to RT and the residuetreated with ethyl acetate (120 ml). Activated charcoal (1.0 g) wasadded and the mixture heated under reflux for 30 min, filtered throughcelite and the filtrate evaporated in vacuo to provide the intermediatephthalimido-acid, a 1:1 mixture of cis/trans isomers, as a pale yellowsolid (45.7 g, 97%).

A solution of this acid in methanol (300 ml) was treated with conc.hydrochloric acid (0.5 ml) and the mixture heated under reflux for 30min, allowed to cool to RT and the solvent evaporated in vacuo. Theresidue was dissolved in ethyl acetate (400 ml) and the solution washedwith 8% sodium bicarbonate (2×100 ml), water (100 ml) and brine (100ml), dried (MgSO₄) and evaporated in vacuo to provide thephthalimodoester as a 1:1 mixture of cis/trans isomers. Separation byflash column chromatography (eluting with 60% ether-pentane) providedthe title compound (9.72 g, 20%) as a white solid.

TLC R_(f) 0.46 (40% pentane-ether)

INTERMEDIATE 25

Trans-3-Phthalimidocyclopent-4,5-enecarboxylic acid

A solution of intermediate 24 (9.72 g, 35.8 mmol) in a mixture of 0.5Nhydrochloric acid (100 ml) and glacial acetic acid (100 ml) was heatedunder reflux for 30 min. The mixture was diluted with water (200 ml) andextracted with ethyl acetate (3×100 ml). The combined extracts werewashed with brine (100 ml), dried (MgSO₄) and evaporated in vacuo toprovide the crude product which was crystallised from ether (7.56 g,82%) as a white solid.

TLC R_(f) 0.48 (1% AcOH-5% MeOH--CH₂ Cl₂)

INTERMEDIATE 26

Cis-3-Phthalimidocyclopentanecarboxylic acid

Intermediate 23 (15.1 g, 58.7 mmol) was hydrogenated at RT andatmospheric pressure over 5% palladium on charcoal (2 g) in ethylacetate (700 ml) overnight. The catalyst was removed by filtrationthrough celite and the filtrate evaporated in vacuo to provide the titlecompound (15 g, 98%) as white solid.

TLC R_(f) 0.37 (1% AcOH-5% MeOH--CH₂ Cl₂)

Similarly prepared was:

INTERMEDIATE 27

Trans-3-Phthalimidocyclopentanecarboxylic acid

From intermediate 25 (7.55 g, 29.3 mmol), as a white solid (7.04 g, 93%)

TLC R_(f) 0.47 (1% AcOH-5% MeOH--CH₂ Cl₂)

INTERMEDIATE 28

Cis-(1,1-Dimethylethyl(3-Phthalimidocyclopentylacetate

A solution of intermediate 26 (5.09 g, 19.06 mmol) in drydichloromethane (60 ml) was treated with oxalyl chloride (3.4 ml, 39.3mmol) then dimethylformamide (1 drop). The mixture was stirred at RT for2 h then the solvent evaporated in vacuo to provide the intermediateacid chloride.

The residue was dissolve in tetrahydrofuran (30 ml) then treated with asolution of diazomethane in ether (200 ml, ca. 80 mmol) at 0° C. Themixture was stirred at RT overnight then evaporated in vacuo to providethe diazoketone as a yellow solid. The diazoketone was dissolved intert-butanol (100 ml) and heated under reflux while a solution of silverbenzoate (438 mg, 1.9 mmol) in triethylamine (5 ml) was added in smallportions over 2 h. The mixture was heated under reflux for a further 1 hthen cooled to RT, filtered through celite and the filtrate evaporatedin vacuo to provide a yellow solid. The residue was dissolved indichloromethane (75 ml) and the solution washed sequentially with 8%sodium bicarbonate (50 ml), water (50 ml) and brine, dried (MgSO₄) andevaporated in vacuo to provide the crude product. Purification by flashcolumn chromatography (eluting with 50% ether-pentane) provided thetitle compound (3.8 g, 60%) as a white solid.

TLC R_(f) 0.52 (50% pentane-ether)

Similarly prepared was:

INTERMEDIATE 29

Trans-1,1-Dimethylethyl) 3-Phthalimidocyclopentylacetate

From intermediate 27 (1.41 g, 5.44 mmol), as a white solid (1.22 g,70%).

TLC R_(f) 0.58 (50% pentane-ether)

INTERMEDIATE 30

Cis-3-Phthalimidocyclopentylacetic acid

A solution of intermediate 28 (1.78 g, 5.4 mmol) in dichloromethane (20ml) was treated with trifluoracetic acid (2.1 ml, 27 mmol) and themixture stirred at RT overnight. The solvent and excess trifluoraceticacid was removed in vacuo to provide the title compound (1.41 g, 96%) asa white solid.

TLC R_(f) 0.32 (30% pentane-ether)

Similarly prepared was:

INTERMEDIATE 31

Trans-3-Phthalimidocyclopentylacetic acid

From intermediate 29 (1.18 g, 3.58 mmol), as a white solid (842 mg,86%).

TLC R_(f) 0.41 (30% pentane-ether)

INTERMEDIATE 32

(RS)2-Bromo-5phthalimidopentanoic acid

Intermediate 19 (5.0 g 20.2 mmol) and thionyl chloride (10 ml) wereheated together at 65° C. for 30 min. N-Bromosuccinimide (5.4 g) andfurther thionyl chloride were added, plus 48% aqueous HBr (1 drop). Thesolution was heated at 60° C. for 10 min then 70° C. for 2 h 15 min.Further N-bromosuccinimide (850 mg) was added and the mixture was heatedat 70° C. for 2 h. Excess thionyl chloride was removed by evaporationunder reduced pressure and the oily residue was diluted with drytetrahydrofuran (200 ml) and water (200 ml). The mixture was thentreated cautiously with solid sodium bicarbonate to pH 7-8 then stirredovernight at RT. Excess tetrahydrofuran was removed in vacuo and theresidue washed with dichloromethane (3×300 ml). The aqueous portion wasthen cautiously acidified to pH 1 using 6M hydrochloric acid andextracted with dichloromethane (4×200 ml). The combined extracts werethen washed with water (2×400 ml) and brine (400 ml), dried (MgSO₄) andevaporated in vacuo to provide the product (4.7 g, 71%) as a fawn solid.

TLC R_(f) 0.47 (EtOAc)

Similarly prepared were:

INTERMEDIATE 33

(RS) 2-Bromo-20(3-phthalimidophenyl)acetic acid

From intermediate 21 as a colourless solid (588 mg, 75%)

TLC R_(f) 0.19 (5% MeOH-0.1% AcOH--CH₂ Cl₂)

INTERMEDIATE 34

Cis-a-Bromo-3-phthalimidocyclopentylacetic acid

From intermediate 30 (1.41 g, 5.16 mmol), as a buff foam (1.59 g, 87%).

TLC R_(f) 0.46 (2% MeOH-ether)

INTERMEDIATE 35

Trans-a-Bromo-3-phthalimidocyclopentylacetic acid

From intermediate 31 (815 mg, 2.98 mmol), as a yellow-brown foam (805mg, 77%)

TLC R_(f) 0.48 (2% MeOH-ether)

INTERMEDIATE 36

(RS)2-Acetylmetcapto-5-phthalimidopentanoic acid

A solution of intermediate 33 (3.0 g, 9.2 mmol) in methanol (30 ml) wastreated with potassium thiolacetate (1.05 g, 9.2 mmol) and the mixturestirred at RT overnight. The mixture was evaporated in vacuo, theresidue dissolved in dichloromethane (100 ml) then the solution washedwith water (2×50 ml), dried (MgSO₄) and evaporated in vacuo to providethe product (2.4 g, 81% as a pale yellow foam.

TLC R_(f) 0.43 (EtOAc)

Similarly prepared were:

INTERMEDIATE 37

(RS) 2-Acetylmercapto-2-(3phthalimido)phenylacetic acid

From intermediate 33 as a colourless solid (722 mg, 100%)

TLC R_(f) 0.15 (5% MeOH-0.1% AcOH--CH₂ Cl₂)

INTERMEDIATE 38

CIS-a-(Acetylmercapto)-3-phthalimidocyclopentylacetic acid

From intermediate 34 (2.01 g, 5.71 mmol), as a brown foam (1.44 g, 73%)

TLC R_(f) 0.42 (Ether)

INTERMEDIATE 39

Trans-a-(Acetylmercapto)-3-phthalimidocyclopentylacetic acid

From intermediate 35 (774 mg, 2.2 mmol), as a beige foam (329 mg, 43%)

TLC R_(f) 0.45 (Ether)

INTERMEDIATE 40

(RS)-N-[2-Bromo-4phthalimidobutanoyl]-L-leucyl-L-phenylanlanine N-methylamide

Intermediate 18 (2.33 g) and thionyl chloride (2.92 ml) were heatedtogether at 65° C. for 30 min. N-Bromosuccinimide (2.51 g) and furtherthionyl chloride were added, plus 48% aqueous HBr (1 drop). The solutionwas heated at 60° C. for 10 min then 70° C. for 2 h 15 min. FurtherN-bromosuccinimide (850 mg) was added and the mixture was heated at 70°C. for 2 h. Excess thionyl chloride was removed by evaporation underreduced pressure and the oily residue was diluted with drydichloromethane (10 ml). A portion of the supernatant (4.0 ml) was addedto a solution of L-leucyl-L-phenylanlanine N-methyl amide (500 mg) andtriethylamine (0.24 ml) in dry dichloromethane (10 ml) at 0° C., andthis mixture was stirred overnight at RT. The mixture was diluted withdichloromethane and washed with saturated aqueous sodium bicarbonatesolution (50 ml), 1N aqueous hydrochloric acid (50 ml), and saturatedbrine (50 ml), then dried (MgSO₄) and evaporated in vacuo to give abrown solid. This material was purified by flash column chromatography(4×18 cm; eluting with 2% methanol-dichloromethane) to give the titlecompound as an off-white solid (530 mg).

TLC R_(f) 0.44 (5% MeOH--CH₂ Cl₂)

Similarly prepared were:

INTERMEDIATE 41

(RS)-N-[2-Bromo-5-phthalimidopentanoyl]-L-leucyl-L-phenylanlanineN-methyl amide

From Intermediate 19 (4.0 g) and L-leucyl-L-phenylalanine N-methyl amide(1.18 g), as a pink solid (1.2 g).

TLC R_(f) 0.34 (5% MeOH--CH₂ Cl₂)

INTERMEDIATE 42

(RS)-N-[2-Bromo-6phthalimidohexanyol]-L-leucyl-L-phenylalanine N-methylamide

From Intermediate 20 (4.0 g) and L-leucyl-L-phenylalanine N-methyl amide(1.18 g), as a near colourless solid (1.0 g).

TLC R_(f) 0.52 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 43

(RS)-2-[(1,1-Dimethylethyl)mercapto]-5-phthalimidopentanoic acid

Tert-butylthiol (11.3 ml, 0.1 mol) was added to a stirred solution ofpotassium tert-butoxide (22.45 g, 0.1 mol) in anhydrous tetrahydrofuran(215 ml) and the mixture stirred at RT for 20 min. A solution ofintermediate 13 (32.6 g, 0.1 mol) in anhydrous tetrahydrofuran (80 ml)was then added and the mixture stirred at RT overnight. Water (300 ml)was added, the mixture acidified to pH 1 with 1N hydrochloric acid andextracted with dichloromethane (3×200 ml). The combined extracts werethen dried (MgSO₄) and evaporated in vacuo to provide a yellow oil.Purification by flash column chromatography (eluting with 5-15%dichloromethane in ethyl acetate) then crystallisation fromdichloromethane/hexane furnished the title compound

(22 g, 66%) as a pale yellow solid.

TLC R_(f) 0.48 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 44

(RS)-N-[2-[(1,1-Dimethylethyl)mercapto]-5-phthalimidopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

A solution of intermediate 43 (8.06 g., 24 mmol) andL-leucyl-L-phenylalanine N-methyl amide (7.0 g, 24 mmol) indichloromethane (250 ml) was treated with N-hydroxybenzotriazole (3.9 g,28.9 mmol) then EDC (5.06 g, 26.4 mmol) and the mixture stirred at RTovernight. The mixture was washed with 1N hydrochloric acid (300 ml) andthe aqueous portion re-extracted with dichloromethane (2×100 ml). Thecombined extracts were washed sequentially with 1N hydrochloric acid(300 ml), 8% sodium bicarbonate (300 ml), water (300 ml) and brine (300ml), dried (MgSO₄) and evaporated in vacuo to provide a pale yellowsolid. Purification by flash column chromatography (eluting with 5%methanol in dichloromethane) provided the title compound (12.8 g, 88% asa near white solid.

TLC R_(f) 0.55 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 45

(RS)-N-[2-[(1,1Dimethylethyl)mercapto]-5-aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

A solution of intermediate 44 (2.56 g, 4.2 mmol) in a mixture oftetrahydrofuran (10 ml) and ethanol (50 ml) was treated with hydrazinehydrate (10 ml, xs) and the mixture heated under reflux for 2 h. Aftercooling to RT water (30 ml) was added, the solvent removed in vacuo, theresidue acidified to pH 1 with 1N hydrochloric acid and washed withdichloromethane (2×100 ml). The aqueous layer was then basified to pH 14with 2M sodium hydroxide and extracted with dichloromethane (2×100 ml).The combined extracts were washed with brine (100 ml), dried (MgSO₄) andevaporated in vacuo to provide a pale yellow solid. Purification byflash column chromatography (eluting with 10-25% methanol indichloromethane) provided the title compound (9.2 g, 91%) as a nearwhite solid.

TLC R_(f) 0.23 (30% MeOH--CH₂ Cl₂)

INTERMEDIATE 46

(RS)-N-[2-[(1,1-Dimethylethyl)mercapto]-5-(acetylamino)pentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

Acetyl chloride (0.5 ml, 7 mmol) was added to a stirred solution ofintermediate 45 (1.14 g, 2.38 mmol) and triethylamine (2 ml, 14.4 mmol)in anhydrous dichloromethane (45 ml), the mixture was then stirred at RTovernight. The mixture was diluted with dichloromethane (75 ml) andwashed successively with 1N hydrochloric acid (100 ml), 8% sodiumbicarbonate (100 ml), water (100 ml), dried (MgSO.sub.) and evaporatedin vacuo to provide a pale yellow solid. Purification by flash columnchromatography (eluting with 5% methanol in dichloromethane) providedthe title compound (1.2 g, 95%) as a near white solid.

TLC R_(f) 0.31 (10% MeOH--CH₂ Cl₂)

Similarly prepared were:

INTERMEDIATE 47

(RS)-N-[2-[(1,1-Dimethylethyl)mercapto]-5-(benzoylamino)pentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 45 (2.86 g, 5.97 mmol) and benzoyl chloride (0.84 ml,7.2 mmol), as a near white solid (3.43 g, 99%).

TLC R_(f) 0.41 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 48

(RS)-N-[2-[(1,1,-Dimethylethyl)mercapto]-5-succinimidopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 45 (1.14 g, 2.38 mmol) and succinic anhydride (0.33ml, 3.3 mmol), as a near white solid (0.54 g, 40%).

TLC R_(f) 0.58 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 49

(RS)-N-[2-[(1,1-Dimethylethyl)mercapto]-5-(methanesulphonyl)aminopentanoyl]-L-leucyl-L-phenylalanine N-methyl amide

From intermediate 45 (2.03 g, 4.24 mmol) and methanesulphonyl chloride(0.35 ml, 4.6 mmol), as a near white solid (1.78 g, 77%).

TLC R_(f) 0.46 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 50

(RS)-N-[2-[(1,1-Dimethylethyl)mercapto]-5-(benzenesulphonyl)aminopentanoyl]-L-leucyl-L-phenylalanine N-methyl amide

From intermediate 45 (2.03 g, 4.24 mmol) and benzenesulphonyl chloride(0.59 ml, 4.6 mmol), as a near white solid (2.18 g, 85%).

TLC R_(f) 0.54 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 51

(RS)-N-[2-[(1,1-Dimethylethyl)mercapto]-5-methoxycarbonyl)aminopentanoyl]-L-leucyl-L-phenylalanine N-methyl amide

From intermediate 45 (200 mg, 0.43 mmol) and methyl chloroformate (0.03ml, 0.41 mmol), as a near white solid (196 mg, 89%).

TLC R_(f) 0.32 (5% MeOH--CH₂ Cl₂)

INTERMEDIATE 52

(RS)-N-[2-[(1,1-Dimethylethyl)mercapto]-5-(benzyloxycarbonyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 45 (200 mg, 0.43 mmol) and benzyl chloroformate (0.06ml, 0.41 mmol), as a near white solid (230 mg, 93%).

TLC R_(f) 0.44 (5% MeOH--CH₂ Cl₂)

INTERMEDIATE 53

(RS)-N-[2-[(1,1-Dimethylethyl)mercapto]-5-(4-pyridylcarbonyl)aminopentanoyl]-L-leucyl-L-phenylalanine N-methyl amide

From intermediate 45 (2.03 g, 4.24 mmol) and isonicotinoyl chloride (834mg, 4.66 mmol), as a near white solid (1.81 g, 77%).

TLC R_(f) 0.22 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 54

(RS)-N-[2-[(1,1-Dimethylethyl)mercapto]-5-(3-pyridylcarbonyl)aminopentanoyl]-L-leucyl-L-phenylalanine N-methyl amide

From intermediate 45 (200 mg, 0.42 mmol) and nicotinoyl chloride (83 mg,0.47 mmol), as a near white solid (130 mg, 56%).

TLC R_(f) 0.26 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 55

(RS)-N-[2-[(1,1-Dimethylethyl)mercapto]-5-(2-pyridylcarbonyl)aminopentanoyl]-L-leucyl-L-phenylalanine N-methyl amide

From intermediate 45 (200 mg, 0.42 mmol) and picolinoyl chloride (83 mg,0.47 mmol), as a near white solid (145 mg, 62%).

TLC R_(f) 0.21 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 56

(RS)-N-[2-[(1,1-Dimethylethyl)mercapto]-5-(2-pyrazinylcarbonyl)aminopentanoyl]-L-leucyl-L-phenylalanine N-methyl amide

From intermediate 45 (200 mg, 0.42 mmol) and pyrazinoyl chloride (85 mg,0.47 mmol), as a near white solid (145 mg, 62%).

TLC R_(f) 0.18 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 57

(RS)-N-[2-[(2-Nitrophenylsulphanyl)mercapto]-5-(acetyl)aminopentanoyl]-L-leucyl-L-phenylalanine N-methyl amide

2-Nitrosulphenyl chloride (3.26 g, 6.26 mmol) was added to a stirredsolution of intermediate 46 (1.23 g, 6.49 mmol) in glacial acetic acid(75 ml) and the mixture stirred at RT overnight. The solvent was removedin vacuo and the residue purified by flash column chromatography(eluting with 5% methanol in dichloromethane) to provide the titlecompound (3.74 g, 97%) as a yellow solid.

TLC R_(f) 0.34 (10% MeOH--CH₂ Cl₂)

Similarly prepared were:

INTERMEDIATE 58

(RS)-N-[2-[(2-Nitrophenylsulphanyl)mercapto]-5-(benzoylamino)pentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 47 (1.08 g, 5.67 mmol), as a yellow solid (3.43 g,99%).

TLC R_(f) 0.54 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 59

(RS)-N-[2-[(2-Nitrophenylsulphanyl)mercapto]-5-succinimidopentanoyl]-L-leucyl-L-phenylalanine N-methyl amide

From intermediate 48 (618 g, 1.1 mmol), as a yellow solid (665 mg, 92%).

TLC R_(f) 0.25 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 60

(RS)-N-[2-[(2-Nitrophenylsulphanyl)mercapto]-5-(methanesulphonyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 49 (1.78 g, 3.27 mmol), as a yellow solid (1.84 g,88%).

TLC R_(f) 0.32 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 61

(RS)-N-[2-[(2-Nitrophenylsulphanyl)mercapto]-5-(benzenesulphonyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 50 (2.25 g, 3.71 mmol), as a yellow solid (1.6 g,68%).

TLC R_(f) 0.37 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 62

(RS)-N-[2-[(2-Nitrophenylsulphanyl)mercapto]-5-(methoxycarbonyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 51 (196 mg, 0.31 mmol), as a yellow solid (86 mg,38%).

TLC R_(f) 0.25 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 63

(RS)-N-[2-[(2-Nitrophenylsulphanyl)mercapto]-5-(benzyloxycarbonyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 52 (229 mg, 0.32 mmol), as a yellow solid (155 mg,60%).

TLC R_(f) 0.37 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 64

(RS)-N-[2-[(2-Nitrophenylsulphanyl)mercapto]-5-4-pyridylcarbonyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 53 (1.92 g, 3.29 mmol), as a yellow solid (1.66 g,74%).

TLC R_(f) 0.29 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 65

(RS)-N-[2-[(2-Nitrophenylsulphanyl)mercapto]-5(3-pyridylcarbonyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 54 (190 mg, 0.33 mmol), as a yellow solid (85 mg,38%).

TLC R_(f) 0.24 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 66

(RS)-N-[2-[(2-Nitrophenylsulphanyl)mercapto]-5(2-pyridylcarbonyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 55 (210 mg, 0.36 mmol), as a yellow solid (120 mg,54%).

TLC R_(f) 0.21 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 67

(RS)-N-[2-[(2-Nitrophenylsulphanyl)mercapto]-5-(2-pyrazinylcarbonyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 56 (300 mg, 0.51 mmol), as a yellow solid (110 mg,42%).

TLC R_(f) 0.18 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 68

(RS)-N-[2-[(2-Nitrophenylsulphanyl)mercapto]-5-aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

TLC R_(f) 0.16 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 69

(R)-N-(Phenylmethoxy)carbonyl-(S-methyl)-L-cysteine

Benzyl chloroformate (11.1 ml, 65 mmol) was added dropwise to a stirredsolution of (S-methyl)-L-cysteine (10 g, 75 mmol) in 2M aqueous sodiumhydroxide (50 ml) and the mixture stirred at RT for 4 h. The mixture wasthen basified to pH14 with further 2M sodium hydroxide and the solutionwashed with ethyl acetate (4×50 ml). The aqueous phase was acidified topH3 with concentrated hydrochloric acid and then extracted with ethylacetate (4×70 ml). The combined extracts were washed with brine, dried(MgSO₄) and evaporated in vacuo to provide the product as a pale yellowoil (16.8 g, 84%)

TLC R_(f) 0.21 (10% MeOH--CHCl₃)

INTERMEDIATE 70

(R)-N-(1,1-Dimethylethoxy)carbonyl-(S-methyl)cysteine

Di-tert-butyldicarbonate (8.88 g, 40.7 mmol) was added to a stirredsolution of S-methyl-L-cysteine methyl-L-cysteine (5 g, 37 mmol) andsodium bicarbonate (7.8 g, 92.5 mmol) in a mixture if water (150 ml) anddioxane (100 ml) at 0° C. The mixture was allowed to warm to RT andstirred overnight, then diluted with water (100 ml), acidified to pH 3using 1N hydrochloric acid then extracted with ethyl acetate (3×100 ml).The combined extracts were washed with brine, dried (MgSO₄) andevaporated in vacuo to provide the title compound as a colourless oil(6.64 g, 76%)

TLC R_(f) 0.55 (MeOH--H₂ O

Similarly prepared was:

INTERMEDIATE 71

(S)-N-(1,1-Dimethylethoxy)carbonyl-propylglycine

From (S)-norvaline (5 g, 42.7 mmol), as a colourless oil (6.3 g, 68%).

TLC R_(f) 0.50 (MeOH--H₂ O)

INTERMEDIATE 72

(S)-N-(1,1-Dimethylethoxy)carbonyl-(O-methyl)serine

Sodium hydride (60% dispersion, 4.3 g, 0.107 mol) was added portionwiseat 0° C. to a stirred solution of(S)-N-(1,1-dimethylethoxy)carbonylserine (10 g, 48.7 mmol) in anhydrousDMF (250 ml). A solution of iodomethane (6.1 ml, 0.1 mol) in anhydrousDMF (10 ml) was then added dropwise. The mixture was allowed to warm toRT and stirred overnight, then diluted with 1N hydrochloric acid to pH3and the solution concentrated in vacuo to ca. 100 ml. The mixture wasdiluted with water (200 ml) and extracted with ethyl acetate (3×100 ml).The combined extracts were washed with brine (100 ml), dried (MgSO₄) andevaporated in vacuo to provide the title compound as a yellow oil (7.48g, 70%)

TLC R_(f) 0.39 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 73

(RS)-N-Benzoyl-[b-(4-pyridyl)]alanine methyl ester

a) 2-(4-Pyridyl)-1-(N-benzoyl)aminoethylene-1-carboxylic acidhydrochloride

4-pyridylcarboxaldehyde (85 g, 0.79 mol) was added to a stirred ice coldsuspension of sodium acetate (16 g, 0.195 mol) and hippuric acid (150 g,0.84 mol) in acetic anhydride (360 ml, 3.8 mol) an exothermic reactionensued and the internal temperature reached 30-40° C. The mixture wascooled to RT then poured into water (1500 ml). The resulting solid wasremoved by filtration washed with water (2×500 ml) to provide theintermediate azlactone (74 g, 37%) as a brown solid The azlactone (70 g,0.28 mol) was dissolved in conc. hydrochloric acid (210 ml) resulting inthe rapid formation of a precipitate which was removed by filtration,washed with acetone (2×500 ml) and dried in vacuo to provide the product(71 g, 78 %) as a pale green solid.

b) (RS)-N-Benzoyl-[b-(4-pyridyl)]alanine hydrochloride

The olefin hydrochloride (70 g, 0.23 mol) was hydrogenated at RT andatmospheric pressure over 5% palladium on carbon (7 g) in water (700 ml)overnight. The catalyst was removed by filtration through celite and thesolvent concentrated to a volume of ca. 100 ml. Acetone (1000 ml) wasadded and the resulting precipitate removed by filtration and dried invacuo to provide the product (53 g, 75%) as an off-white solid.

c) (RS)-N-Benzoyl-[b-(4-pyridyl)]alanine methyl ester

Thionyl chloride (15 ml, 0.21 mol) was added to an ice cold solution ofthe acid (53 g, 0.17 mol) in methanol (200 ml). The mixture was allowedto warm to RT over 30 min before the solvent was removed in vacuoproviding an off-white semi-solid. The residue was suspended in 8%sodium bicarbonate (500 ml) and the product extracted into ethyl acetate(4×400 ml). The combined organic extracts were then washed with brine(1000 ml), dried (MgSO₄) and evaporated in vacuo providing the titlecompound (46 g, 94%) as an off-white solid.

C₁₆ H₁₆ N₂ O₃ [284.3]; [MH⁺ 285]

Similarly prepared were:

INTERMEDIATE 74

(RS)-N-Benzoyl-[b-(3-pyridyl)]alanine methyl ester

In three steps from pyridine-3-carboxaldehyde as a white solid.

C₁₆ H₁₆ N₂ O₃ [284.3]; [MH⁺ 285]

INTERMEDIATE 75

(RS)-N-Benzoyl-2-methoxyalanine methyl ester

In three steps from 2-methoxybenzaldehyde as a white solid

C₁₇ H₁₉ N₂ O₄ [301.3]; [MH⁺ 302]

INTERMEDIATE 76

(S)-N-(1,1-Dimethylethoxy)carbonyl-[b-(4-pyridyl)]alanine

Intermediate 73 (27 g, 0.104 mol) was dissolved in hot acetone (50 ml)and the solution added to 0.03M potassium dihydrogen phosphate (500 ml)at pH7.2.

Alcalase (1 ml) was then added to the vigorously stirred solution andthe pH maintained at 7.2 by the addition of 1M sodium hydroxide (ca. 20ml) over 30 min. Undissolved ester was filtered off and the filtratewashed with ethyl acetate (4×250 ml). The solvent was then evaporated invacuo, the residue dissolved in 6N hydrochloric acid (100 ml) thenheated under reflux for 5 h. After cooling to RT the precipitatedbenzoic acid was removed by filtration and the filtrate washed withethyl acetate (2×100 ml). The aqueous portion was then evaporated invacuo to provide the optically pure amino-acid as its dihydrochloridesalt (9.48 g, 81%).

The dihydrochloride salt was dissolved in water (150 ml) and treatedwith Amberlite IRA-67 ion exchange resin to pH 8. The resin was removedby filtration through celite and the solution evaporated in vacuo toprovide the free amino-acid as a white solid (6.7 g, 100%).

Di-tert-butyldicarbonate (13 g, 60 mmol) was added to a stirredsuspension of the amino-acid in a mixture of tert-butanol (100 ml) andwater (50 ml) while the pH was maintained at 8.5 by the addition of 1Msodium hydroxide (55 ml) over 40 min. Water (50 ml) was added, themixture washed with heptane (2×100 ml) then acidified to pH 3.5 by theaddition of solid potassium hydrogen sulphate. The mixture was thenevaporated to dryness and the residue triturated with methanol (3×150ml) to extract the product. Evaporation of the solvent in vacuo providedthe title compound as a white solid which was recrystallised fromaqueous ethanol to optical purity (3.27 g, 61%).

[a]_(D) +24.3° (c=1, Trifluoroacetic acid)

Similarly prepared were:

INTERMEDIATE 77

(S)-(1,1-Dimethylethoxy)carbonyl-[b-(3-pyridyl)]alanine

From intermediate 74 as a white solid

[a]_(D) +16.1° (c=1, Trifluoroacetic acid)

INTERMEDIATE 78

(S)-(1,1-Dimethylethoxy)carbonyl-2-methoxyphenylalanine

From intermediate 75 as a white solid (11.1 g, 97%)

[a]_(D) -15.2° (c=1, MeOH)

INTERMEDIATE 79

(RS)-N-Acetyl-[b-(2-pyridyl)]alanine

A solution of sodium methoxide (65.8 g, 1.22 mol) in methanol (300 ml)was treated portionwise with diethylacetamidomalonate (132.3 g, 0.61mol) maintaining a temperature of ca. 45° C. The mixture was then heatedunder reflux for 15 min. The mixture was cooled to 50° C. then treatedslowly with a suspension of 2-chloromethylpyridine hydrochloride (100 g,0.61 mol), the pink suspension was then heated under reflux for afurther 6 h. Water (500 ml) was added followed by 10M sodium hydroxide(122 ml, 1.22 mol) and the pH was maintained at ca. 11 while heating themixture at 70° C. overnight. The mixture was cooled to RT and themethanol removed in vacuo. The aqueous residue was washed with ethylacetate (2×500 ml), then acidified to pH 5 and further washed with ethylacetate (2×500 ml), before evaporating in vacuo to provide a semi-solid.The residue was triturated with hot ethanol (500 ml) and the sodiumchloride removed by filtration. The filtrate was then evaporated invacuo and the residue crystallised from methanol-ethyl acetate toprovide the title compound (70 g, 66%) as a yellow solid.

TLC R_(f) 0.5 [n-BuOH-AcOH-pyridine-H₂ O (15-3-10-12)]

Similarly prepared were:

INTERMEDIATE 80

(RS)-N-Acetyl-[b-(4-thiazolyl)]alanine

From 4-(chloromethyl)thiazole, as a white foam (6.7 g, 73%).

TLC Rf 0.37 (1% AcOH-20% Hexane-EtOAc)

INTERMEDIATE 81

(S)-(1,1-Dimethylethoxy)carbonyl-[b-(2-pyridyl)]alanine

A suspension of intermediate 79 (65 g, 0.31 mol) in aqueous potassiumdihydrogen orthophosphate (10 mM, 650 ml) was warmed to 40° C. and theresulting mixture (pH 4) basified to pH 8 with 10M sodium hydroxide (10ml) forming a solution. Acylase 30,000 (1.3 g) was added and the mixtureincubated at 40° C. overnight. The resulting suspension was cooled toRT, the solid removed by filtration, then the filtrate acidified to pH 1with 2N hydrochloric acid and washed with ethyl acetate (2×300 ml). Theaqueous portion was evaporated in vacuo to provide a semi-solid whichwas triturated with hot methanol (300 ml) and the solid removed byfiltration. To the cooled solution was then added 5M sodium hydroxide topH 10, followed by di-tert-butyldicarbonate (28.6 g, 0.131 mol), themixture was maintained at pH 10 by the addition of 5M sodium hydroxide(44 ml) over a period of 6 h. The methanol was then removed in vacuo,the aqueous residue washed with ether (2×500 ml) then acidified to pH 3with 1M potassium hydrogen sulphate. The mixture was extracted withethyl acetate (4×500 ml), the combined extracts washed with brine (500ml), dried (MgSO₄) and evaporated in vacuo to provide the crude productwhich was crystallised from ethyl acetate as a white solid (11.6 g,23%).

[a]_(D) -16.0° (c=1, MeOH)

Similarly prepared were:

INTERMEDIATE 82

(S)-N-(1,1-Dimethylethoxy)carbonyl-[b-(4-thiazolyl)]alanine

From intermediate 80, as a white solid (11 g, 94%)

HPLC (Chirex (D)-penicillamine; 2 mM CuSO₄ ; 0.7 ml/min; RT=3.25 min;99% ee.

INTERMEDIATE 83

(S)-N-(1,1-Dimethylethoxy)carbonyl-[b-(2-pyridyl)]alanine N-methyl amide

DCC (4.07 g, 19.7 mmol) was added to an ice cooled solution ofintermediate 81 (5.0 g, 18.8 mmol) and N-hydroxysuccinimide (2.27 g,19.7 mmol) in dry tetrahydrofuran (200 ml). After stirring at RT for 3 hthe mixture was treated with 40% w/v aqueous methylamine (6.8 ml, 79mmol) and stirred for a further 2 h. The precipitated solid was removedby filtration, the filtrate evaporated in vacuo and the residuedissolved in dichloromethane (100 ml). The solution was washedsequentially with water (2×100 ml), 8% sodium bicarbonate (2×100 ml) andbrine (100 ml), dried (MgSO₄) and evaporated in vacuo to provide thecrude product.

Purification by column chromatography eluting with 2-5%methanol/dichloromethane provided the title compound (2.7 g, 51%) as ared foam.

TLC R_(f) 0.38 (3% MeOH--CH₂ Cl₂)

Similarly prepared were:

INTERMEDIATE 84

(S)-N-(1,1-Dimethylethoxy)carbonyl-L-[b-(4-thiazolyl(]alanine N-methylamide

From intermediate 82, as a white solid (3.0 g, 64%).

TLC R_(f) 0.46 (5% MeOH--CH₂ Cl₂)

INTERMEDIATE 85

(R)-N-(1,1-Dimethylethoxy)carbonylpenicillamine N-methyl amide

A solution of (R)-N-(1,1-dimethylethoxy)carbonylpenicillamine (14 g,56.1 mmol), N-hydroxybenzotriazole (7.6 g, 56.1 mmol), methylaminehydrochloride (18.9 g, 280 mmol), N-methylmorpholine (34 ml, 308 mmol,4-dimethylaminopyridine (685 mg, 5.6 mmol) and EDC (11.8 g, 62 mmol) inanhydrous dimethylformamide (300 ml) was stirred at RT overnight. Themixture was poured into 10% w/v citric acid (750 ml) and extracted withether (3×500 ml). The combined extracts were then washed with 8% sodiumbicarbonate (2×500 ml) and brine (500 ml), dried (MgSO₄) and evaporatedin vacuo to provide the title compound (12.3 g, 83%)

TLC R_(f) 0.47 (50% hexane-EtOAc)

Similarly prepared was:

INTERMEDIATE 86

(1,1-Dimethylethoxy)carbonyl-5-methyl-L-glutamic acid N-methyl amide

From (1,1-dimethylethoxy)carbonyl-5-methyl-L-glutamic acid, as a whitesolid (5.4 g, 94%).

TLC R_(f) 0.21 (2% MeOH--CH₂ Cl₂)

INTERMEDIATE 87

(S)-N_(d) -(1,1-Dimethylethoxy)carbonyl-N_(a)-(benzyloxy)carbonylornithine N-methyl amide

From (S)-N_(d) -(1,1-dimethylethoxy)carbonyl-N_(a)-(benzyloxy)carbonylornithine (11.5 g, 31.4 mmol), as a white solid(11.0 g, 94%).

TLC R_(f) 0.65 (3% MeOH--CH₂ Cl₂)

INTERMEDIATE 88

(S)-N-(1,1-Dimethylethoxy)carbonyl-2-methoxyphenylalanine N-methyl amide

From intermediate 78 (500 mg, 1.69 mmol), as a white solid (380 mg,73%).

TLC R_(f) 0.55 (5% MeOH--CH₂ Cl₂)

INTERMEDIATE 89

(S)-N-(1,1-Dimethylethoxy)carbonyl-b-(3-pyridyl)alanine N-methyl amide

From intermediate 77 (2.0 g, 7.5 mmol), as a white solid (1.0 g, 48%).

TLC R_(f) 0.42 (5% MeOH--CH₂ Cl₂)

INTERMEDIATE 90

(S)-N-(1,1-Dimethylethoxy)carbonyl-b-(4-pyridyl)alanine N-methyl amide

From intermediate 76 (2.0 g, 7.5 mmol), as a white solid (1.7 g, 81%).

TLC R_(f) 0.38 (5% MeOH--CH₂ Cl₂)

INTERMEDIATE 91

(R)-N-(1,1-Dimethylethoxy)carbonyl-(S-methyl)penicillamine N-methylamide

A solution of iodomethane (0.59 ml, 9.55 mmol) in methanol (5 ml) wasadded dropwise at 0° C. to a stirred solution of intermediate 85 (500mg, 1.51 mmol) in a mixture of 2M sodium hydroxide (5 ml) and methanol(15 ml). The mixture was stirred at RT overnight, then the methanolremoved in vacuo, the residue diluted with water (50 ml) and extractedwith ether (3×50 ml). The combined extracts were washed with brine,dried (MgSO₄) and evaporated in vacuo to provide the title compound as acolourless oil (540 mg, 99%).

TLC R_(f) 0.47 (50% hexane-EtOAc)

INTERMEDIATE 92

(R)-N-(1,1-Dimethylethoxy)carbonyl-2,2-dimethyl-2-methanesulphonylalanine N-methyl amide

A suspension of Oxone (1.02 g, 1.66 mmol) in water (5 ml) was added to astirred solution of intermediate 91 (153 mg, 0.55 mmol) in methanol (5ml) at 0° C., the mixture was allowed to warm to RT and stirred for 4 h.The mixture was diluted with water (50 ml) and extracted with ethylacetate (3×50 ml). The combined extracts were washed with brine (50 ml),dried (MgSO₄) and evaporated in vacuo to provide the title compound as acolourless oil (101 mg, 59%).

TLC R_(f) 0.35 (50% hexane-EtOAc)

INTERMEDIATE 93

(R)-N-(1,1-Dimethylethoxy)carbonyl-2,2-dimethyl-2-methanesulphinylalanine N-methyl amide

A solution of 3-chloroperbenzoic acid (670 mg, 2.89 mmol) indichloromethane (25 ml) was added to a stirred solution of intermediate91 (800 mg, 2.89 mmol) in dichloromethane (25 ml) at 0° C., the mixturewas allowed to warm to RT and stirred overnight. The mixture was washedsequentially with 10% w/v sodium sulphite (2×50 ml), 8% sodiumbicarbonate (2×50 ml) and brine (50 ml), dried (MgSO₄) and evaporated invacuo to provide the crude product as a colourless oil. Purification byflash column chromatography (eluting with 10% methanol-dichloromethane)provided the title compound as a colourless oil (470 mg, 55%).

TLC R_(f) 0.05 (50% hexane-EtOAc)

INTERMEDIATE 94

(R)-N-2,2-Dimethyl-2-methanesulphinylalanine N-methyl amidehydrochloride

A solution of intermediate 93 (470 mg, 1.61 mmol) in a mixture ofdioxane (25 ml) and 2M hydrochloric acid (25 ml) was stirred at RTovernight. The solvent was evaporated to dryness in vacuo and freezedried overnight to provide the title compound (370 mg, 100%) as acolourless gum.

TLC R_(f) 0.06 (1% NEt₃ -10% MeOH--CH₂ Cl₂)

Similarly prepared were:

INTERMEDIATE 95

(R)-N-2,2-Dimethyl-2-methanesulphonylalanine N-methyl amidehydrochloride

From intermediate 92 (300 mg, 0.97 mmol), as a colourless foam (210 mg,100%).

TLC R_(f) 0.10 (1% NEt₃ -10% MeOH--CH₂ Cl₂)

INTERMEDIATE 96

(R)-Penicillamine N-methyl amide hydrochloride

From intermediate 85 (300 mg, 1.14 mmol), as a colourless foam (230 mg,100%).

TLC R_(f) 0.13 (1% NEt₃ -10% MeOH--CH₂ Cl₂)

INTERMEDIATE 97

(S)-b-(3-Pyridyl)alanine N-methyl amide dihydrochloride

From intermediate 89 (1.2 g, 4.29 mmol), as a white solid (1.1 g, 100%).

TLC R_(f) 0.05 (1% NEt₃ -10% MeOH--CH₂ Cl₂)

INTERMEDIATE 98

(S)-b-(4-Pyridyl)alanine N-methyl amide dihydrochloride

From intermediate 90 (1.6 g, 5.73 mmol), as a white solid (1.45 g,100%).

TLC R_(f) 0.08 (1% NEt₃ -10% MeOH--CH₂ Cl₂)

INTERMEDIATE 99

(S)-b-(2-Pyridyl)alanine N-methyl amide dihydrochloride

From intermediate 83 (1.4 g, 5.01 mmol), as a white solid (1.27 g,100%).

TLC R_(f) 0.13 (1% NEt₃ -10% MeOH--CH₂ Cl₂)

INTERMEDIATE 100

(S)-b-(4-Thiazolyl)alanine N-methyl amide hydrochloride

From intermediate 84 (1.0 g, 3.5 mmol), as a white solid (730 mg, 94%).

TLC R_(f) 0.21 (1% NEt₃ -10% MeOH--CH₂ Cl₂)

INTERMEDIATE 101

(R)-(S-Methyl)penicillamine N-methyl amide trifluoroacetate

A solution of intermediate 91 (200 mg, 0.72 mmol) in dichloromethane (4ml) containing trifluoroacetic acid (2 ml) was stirred at RT overnight.The solvent was removed in vacuo and any excess trifluoroacetic acidremoved by azeotropic distillation with heptane (3×20 ml) to provide thetitle compound (196 mg, 94%) as a colourless foam.

TLC R_(f) 0.32 (1% NEt₃ -10% MeOH--CH₂ Cl₂)

Similarly prepared were:

INTERMEDIATE 102

(S)-2-(Methoxyphenyl)alanine N-methyl amide trifluoroacetate

From intermediate 88 (200 mg, 0.65 mmol), as a pale yellow foam (209 mg,100%).

TLC R_(f) 0.25 (1% NEt₃ -10% MeOH--CH₂ Cl₂)

INTERMEDIATE 103

(S)-5-(Methyl-glutamic acid N-methyl amide trifluoroacetate

From intermediate 86 (707 mg, 2.58 mmol), as a white solid (728 mg,98%).

TLC R_(f) 0.37 (1% NEt₃ -10% MeOH--CH₂ Cl₂)

INTERMEDIATE 104

(S)-N_(d) -(1,1-Dimethylethoxy)carbonylornithine N-methyl amide

Intermediate 87 (11 g, 29 mmol) was hydrogenated at RT and atmosphericpressure over 10% palladium on carbon (1 g) in ethanol overnight. Thecatalyst was removed by filtration through hyflo and the filtrateevaporated in vacuo to provide the title compound (2.6 g, 36%) as acolourless oil.

TLC R_(f) 0.37 (5% MeOH--CH₂ Cl₂)

INTERMEDIATE 105

N-(Phenylmethoxy)carbonyl-(S-methyl-L-cysteinyl-L-phenylalanine N-methylamide

EDC (10.5 g, 55 mmol) was added to a stirred solution of L-phenylalanineN-methyl amide (8.9 g, 50 mmol), intermediate 69 (13.8 g, 50 mmol) andN-hydroxybenzotriazole (8.1 g, 60 mmol) in dry tetrahydrofuran (100 ml).The mixture was stirred at RT overnight. The mixture was treated with 1Mhydrochloric acid (300 ml) then extracted with ethyl acetate (4×200 ml).The combined organic extracts were washed with 8% sodium bicarbonate(2×200 ml), water (200 ml) and brine (200 ml), dried (MgSO₄) andevaporating in vacuo to provide the product as a white solid (16.5 g,75%)

TLC R_(f) 0.47 (10% MeOH--CHCl₃)

INTERMEDIATE 106

N-(1,1-Dimethylethoxy)carbonyl-(S)-propylglycinyl-L-phenylalanineN-methyl amide

From intermediate 71 (5 g, 23 mmol) and L-phenylalanine N-methyl amide(4.1 g, 23 mmol), as a white solid (7.72 mg, 89%).

TLC R_(f) 0.48 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 107

N-(1,1-Dimethylethoxy)carbonyl-L-leucyl-L-tert-leucine N-methyl amide

From N-(1,1dimethylethoxy)carbonyl-L-leucine (13.07 g, 56 mmol) andL-tert-leucine N-methyl amide (8.11 g, 56 mmol), as a white solid (15.77g, 79%).

TLC R_(f) 0.25 (5% MeOH--CH₂ Cl₂)

INTERMEDIATE 108

N-(1,1-Dimethylethoxy)carbonyl-(S)-(S-methyl)cysteinyl-L-tert-leucineN-methyl amide

From intermediate 70 (6.64 g, 28 mmol) and L-tert-leucine N-methyl amide(4.07 g, 28 mmol), as a white solid (4.26 g, 42%).

TLC R_(f) 0.45 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 109

N-(1,1-Dimethylethoxy)carbonyl-(S)-(O-methyl)serinyl-L-tert-leucineN-methyl amide

From intermediate 72 (7.48 g, 34 mmol) and L-tert-leucine N-methyl amide(4.92 g, 34 mmol), as a white solid (4.26 g, 42%).

TLC R_(f) 0.40 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 110

N-(Phenylmethoxy)carbonyl-L-valinyl-N_(d) -(1,1-dimethylethoxy)carbonyl-L-ornithine N-methyl amide

From intermediate 104 (2.6 g, 10.6 mmol) andN-(Phenylmethoxy)carbonyl-L-valine (2.82 g, 11.2 mmol), as a white solid(3.0 g, 61%).

TLC R_(f) 0.32 (5% MeOH--CH₂ Cl₂)

INTERMEDIATE 111

(S-Methyl)-L-cysteinyl-L-phenylalanine N-methyl amide

A solution of intermediate 105 (2.0 g, 4.65 mmol) in dichloromethane (10ml) was treated with 25% hydrobromic acid in acetic acid (18.6 ml) andthe mixture stirred at RT for 1 h. Water (10 ml) was added and themixture washed with dichloromethane (3×15 ml). The aqueous phase wasthen basified to pH14 with 5M sodium hydroxide then extracted withdichloromethane (4×30 ml). The combined organic extracts were thenwashed with brine (30 ml), dried (MgSO₄) and evaporated in vacuo toprovide the product as a white solid (1.22 mg, 88%).

TLC R_(f) 0.31 (10% MeOH--CHCl₃)

INTERMEDIATE 112

L-Valinyl-N_(d) -(1,1-dimethylethoxy)carbonyl-L-ornithine N-methylamide

Intermediate 110 (3.0 g, 6.5 mmol) was hydrogenated at RT andatmospheric pressure over 10% palladium on carbon (300 mg) in ethanol(100 ml) overnight. The catalyst was removed by filtration through hyfloand the filtrate evaporated in vacuo to provide the title compound (2.2g, 99%) as a white solid.

TLC R_(f) 0.26 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 113

(S)-Propylglycinyl-L-phenylalanine N-methyl amide

A solution of intermediate 106 (5.36 g, 14.2 mmol) in a mixture ofdioxane (250 ml) and 2M hydrochloric acid (250 ml) was stirred at RTovernight. The solvent was evaporated to dryness in vacuo to provide awhite solid. The residue was dissolved in water (200 ml) washed withdichloromethane (3×100 ml) then basified to pH 10 with 2M sodiumhydroxide and extracted with dichloromethane (4×100 ml). The combinedextracts were washed with brine (100 ml), dried (MgSO₄) and evaporatedin vacuo to provide the title compound as a white solid (1.45 g, 37 %).

TLC R_(f) 0.29 (10% MeOH--CH₂ Cl₂)

Similarly prepared were:

INTERMEDIATE 114

(S)-(S-Methyl)cysteinyl-L-tert-leucine N-methyl amide

From intermediate 108, as a white solid (3.5 g, 97%).

TLC R_(f) 0.45 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 115

(S)-(O-Methyl)serinyl-L-tert-leucine N-methyl amide

From intermediate 109, as a white solid (2.7 g, 98%).

TLC R_(f) 0.40 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 116

L-Leucyl-L-tert-leucine N-methyl amide

From intermediate 107, as a white solid (11.2 g, 99%).

TLC R_(f) 0.57 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 117

(R)-2-Bromo-5-phthalimidopentanoic acid

A solution of D-ornithine hydrochloride (35 g, 0.208 mol) in water (350ml) was treated with copper (II) sulphate (16.6 g, 0.104 mol). 5MPotassium hydroxide (ca. 40 ml) was added to pH 3 thenN-carboethoxyphthalimide (45.5 g, 0.208 mol) was added and the pHmaintained at 9-10 by the addition of 5M potassium hydroxide (ca. 55ml). After 2 h, 48% hydrobromic acid was added to pH 0.4 (ca. 77 ml) andany resulting precipitate removed by filtration. The filtrate was cooledto <5° C., then treated with further hydrobromic acid (152 ml) andpotassium bromide (59 g, 0.5 mol). The mixture was then treated dropwiseover 45 min with a solution of sodium nitrite (28.6 g, 0.41 mol) inwater (275 ml) whilst maintaining a temperature of <5° C. The mixturewas then stirred at <5° C. overnight. The resulting precipitate was thenremoved by filtration, dissolved in ethyl acetate (400 ml) and thesolution washed with water (2×200 ml) and brine (200 ml), dried (MgSO₄)and evaporated in vacuo to provide the title compound (40.6 g, 47%) as acream solid.

TLC R_(f) 0.80 (10% MeOH--CH₂ Cl₂)

Similarly prepared was:

INTERMEDIATE 118

(S)-2-Bromo-5phthalimidopentanoic acid

From L-ornithine hydrochloride (20 g, 0.118 mol), as a cream solid (22g, 57%).

TLC R_(f) 0.80 (10% MeOH--CH₂ Cl₂)

INTERMEDIATE 119

(S)-2-Acetylmercapto-5-phthalimidopentanoic acid

Was prepared by the previously described procedure from intermediate 117(39.9 g, 0.11 mol), as a pale orange oil (35.6 g, 99%).

TLC R_(f) 0.24 (50% Heptane-EtOAc)

INTERMEDIATE 120

(S)-2-Acetylmercapto-5-phthalimidopentanoic acid

Was prepared by the previously described procedure from intermediate 118(20 g, 56 mmol), as a pale orange oil (17.7 g, 99%).

TLC R_(f) 0.24 (50% Heptane-EtOAc)

INTERMEDIATE 121

(RS)-2-(Acetylmercapto)-5-phthalimidopentanoyl-L-leucine1,1-dimethylethyl ester

EDC (3.64 g, 19 mmol) was added to a stirred mixture of L-leucine1,1-dimethylethyl ester (3.93 g, 17.6 mmol), N-hydroxybenzotriazole(2.62 g, 19.4 mmol), triethylamine (2.51 ml, 18 mmol) and intermediate15 (5.94 g, 18.5 mmol), triethylamine (200 ml). The mixture was stirredovernight then the solvent removed in vacuo and the residue partitionedbetween water (100 ml) and ethyl acetate (100 ml). The aqueous portionwas then extracted with ethyl acetate (2×50 ml), the combined extractswashed with water (2×100 ml) and brine (100 ml), dried (MgSO₄) andevaporated in vacuo to a colourless oil.

Purification by column chromatography eluting with hexane/ethyl acetate(2:1) provided the title compound (6.6 g, 77%) as a white solid, a 1:1mixture of diastereoisomers.

TLC R_(f) 0.42 (EtOAc/Hexane (1:1))

INTERMEDIATE 122

(RS)-2-(Acetylmercapto)-5-phthalimidopentanoyl-L-leucine

Trifluoroacetic acid (9.0 ml, 115 mmol) was added to a stirred solutionof intermediate 121 (3.0 g, 6.1 mmol) in dry dichloromethane (40 ml) andthe mixture stirred at RT overnight. The mixture was concentrated invacuo and the excess trifluoroacetic acid removed by azeotroping withheptane to provide the title compound (2.48 g, 94%) as a colourlessfoam, a 1:1 mixture of diastereoisomers.

TLC R_(f) 0.42 (EtOAc/Hexane (3:2))

INTERMEDIATE 123

(RS)-2-Acetylmercapto)-4-succinimidobutanoic acid

Was prepared by the procedure previously described for intermediate 36

TLC R_(f) 0.38 (HOAc/EtOAc/Hexane (0.1:1:1))

INTERMEDIATE 124

(S-Methyl)-L-cysteinyl-L-tryptophan N-methyl amide

Was prepared by the procedure previously described for intermediate 111

TLC R_(f) 0.45 (EtOAc/Hexane (1:1))

INTERMEDIATE 125

(RS)-2-[(1,1-Dimethylethyl)mercapto]-3-phthalimidopropananoic acid

Was prepared by the procedure previously described for intermediate 43.

TLC R_(f) 0.48 (HOAc/EtOAc/Hexane (0.1:1:1))

EXAMPLE 1

(RS)-N-[2,3-Bis-acetylmercaptopropanoyl]-L-leucyl-L-phenylalanine Nmethyl amide

EDC (198 mg) was added to a solution of intermediate 14 (209 mg),L-leucyl-L-phenylalanine N-methyl amide (274 mg), andN-hydroxybenzotriazole hydrate (153 mg) in dry THF (10 ml) at 0° C., andthe mixture was stirred at that temperature until TLC analysis (5%MeOH--CH₂ Cl₂) indicated a complete consumption of starting materials(72 h). The solvent was removed by evaporation and the residue waspartitioned between 1N hydrochloric acid (35 ml) and ethyl acetate (50ml). The organic layer was separated, washed with aqueous sodiumbicarbonate solution (2×200 ml) and brine (2×20 ml), then dried (MgSO₄)and evaporated in vacuo to give the crude product. Purification by flashcolumn chromatography (eluting with 2-5% methanol-dichloromethane) gavethe title compound as a colourless solid (264 mg).

TLC R_(f) 0.25 (2% MeOH--CH₂ Cl₂)

Similarly prepared were:

EXAMPLE 2

(RS)-N-[(2,4-Bis-acetylmercapto)butanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 15 (870 mg, 3.7 mmol) and L-leucyl-L-phenylalanineN-methyl amide (1.07 g, 3.7 mmol), as a white solid (1.4 g, 74%).

C₂₄ H₃₅ N₃ O₅ S₂ [509.7]; [MH⁺ =510]

EXAMPLE 3

(RS)-N-[(2,5-Bis-acetylmercapto)pentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 16 (906 mg, 3.6 mmol) and L-leucyl-L-phenylalanineN-methyl amide (1.06 g, 3.6 mmol), as a white solid (1.5 g, 79%).

C₂₅ H₃₇ N₃ O₅ S₂ [523.7]; [MH⁺ =524]

EXAMPLE 4

(RS)-N-[(2,6-Bis-acetylmercapto)hexanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 17 (895 mg, 3.4 mmol) and L-leucyl-L-phenylalanineN-methyl amide (991 mg, 3.4 mmol), as a white solid (1.4 g, 74%).

C₂₆ H₃₉ N₃ O₅ S₂ [537.75]; [MH⁺ =538]

EXAMPLE 5

(RS)-N-[2-Acetylmercapto-3-methoxycarbonylpropranoyl]-L-leucyl-L-phenylalanineN-methyl amide

From Intermediate 4 (0.26 g, as colourless solid (0.33 g).

C₂₃ H₃₃ N₃ O₆ S [479.6]; [MH⁺ =480]

EXAMPLE 6

(RS)-N-[2-Acetylmercapto-4-methoxycarbonylbutanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From Intermediate 5 (0.40 g, as colourless solid (0.67 g).

C₂₄ H₃₅ N₃ O₆ S [493.6]; [MH⁺ =494]

EXAMPLE 7

(RS)-N-[2-Acetylmercapto-5-methoxycarbonylpentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From Intermediate 6 (0.9 g, as colourless solid (1.1 g).

C₂₅ H₃₇ N₃ O₆ S [507.6]; [MH⁺ =508]

EXAMPLE 8

(RS)-N-[2-Acetylmercapto-6-methoxycarbonylhexanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From Intermediate 7 (0.95 g, as colourless solid (1.0 g).

C₂₆ H₃₉ N₃ O₆ S [521.6]; [MH⁺ =522]

EXAMPLE 9

(RS)-N-[2-Acetylmercapto-5-phthalimidopentanoyl]-L-leucyl-L-tryptophanN-methyl amide

From Intermediate 36 (730 mg, and L-leucyl-L-tryptophan N-methyl amide(700 mg), as a pale yellow foam (1.0 g, 74%).

C₃₃ H₃₉ N₅ O₆ S [633.7]; [MH⁺ =634]

EXAMPLE 10

(RS)-N-[2-Acetylmercapto-6-methoxycarbonylhexanoyl]-L-leucyl-L-tryptophanN-methyl amide

From Intermediate 7 (375 mg, and L-leucyl-L-tryptophan N-methyl amide(500 mg), as a pale yellow foam (440 mg, 52%).

C₂₈ H₄₀ N₄ O₆ S [560.7]; [MH⁺ =561]

EXAMPLE 11

(RS)-N-[2-Acetylmercapto-5-phthalimidopentanoyl]-L-valinyl-L-phenylalanineN-methyl amide

From Intermediate 36 (232 mg, and L-valinyl-L-phenylalanine N-methylamide (200 mg), as a white solid (230 mg, 55%).

C₃₀ H₃₆ N₄ O₆ S [580.7]; [MH⁺ =581]

EXAMPLE 12

(RS)-N-[2-Acetylmercapto-6-methoxycarbonylhexanoyl]-L-valinyl-L-phenylalanineN-methyl amide

From Intermediate 7 (179 mg, and L-valinyl-L-phenylalanine N-methylamide (200 mg), as a white solid (200 mg, 56%).

C₂₅ H₃₇ N₃ O₆ S [507.6]; [MH⁺ =508]

EXAMPLE 13

(RS)-N-[2-Acetylmercapto-2-(3-phthalimidophenyl)acetyl]-L-leucyl-L-phenylalanineN-methyl amide

From Intermediate 37 (690 mg, and L-leucyl-L-phenylalanine N-methylamide (489 mg), as a white solid (927 mg, 88%).

C₃₄ H₃₆ N₄ O₆ S [628.8]; [MH⁺ =629]

EXAMPLE 14

N-[2-(Acetylmercapto)-2-[3-cis-phthalimidocyclopentyl]acetyl-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 38 (140 mg, 0.4 mmol) and L-leucyl-L-phenylalanineN-methyl amide (117 mg, 0.4 mmol), as a tan foam (197 mg, 79%), a1:1:1:1 of the four expected diastereoisomers.

C₃₃ H₄₀ N₄ O₆ S [620.8]; [MH⁺ =621]

EXAMPLE 15

N-[2-(acetylmercapto)-2-[3-trans-phthalimidocyclopentyl]acetyl-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 39 (235 mg, 0.7 mmol) and L-leucyl-L-phenylalanineN-methyl amide (197 mg, 0.7 mmol), as abrown foam (358 mg, 85%), a1:1:1:1 of the four expected diastereoisomers.

C₃₃ H₄₀ N₄ O₆ S [620.8]; [MH⁺ =621]

EXAMPLE 16

(S)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-_(L)-leucyl-(S)-tert-leucine N-methyl amide

From intermediate 119 (13.8g, 43 mmol) and intermediate 116 (11.2g, 44mmol), as a white solid (11.6 g, 48%).

C₂₈ H₄₀ N₄ O₆ S [560.7]; [MH⁺ =561]

EXAMPLE 17

(S)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

From intermediate 119 (10 g, 31 mmol) and _(L) -leucyl-_(L)-phenylalanine N-methyl amide (9.0 g, 31 mmol), as a white solid (10.5g, 57%).

C₃₁ H₃₈ N₄ O₆ S [594.7]; [MH⁺ =595]

EXAMPLE 18

(R)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

From intermediate 120 (1.0 g, 3.1 mmol) and _(L) -leucyl-_(L)-phenylalanine N-methyl amide (900 mg, 3.1 mmol), as a white solid (885mg, 48%).

C₃₁ H₃₈ N₄ O₆ S [594.7]; [MH⁺ =595]

EXAMPLE 19

(S)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-_(L) -leucyl-_(L)-tryptophan N-methyl amide

From intermediate 119 (411 mg, 1.28 mmol) and _(L) -leucyl-_(L)-trypotophan N-methyl amide (423 mg, 1.28 mmol), as a white solid (330mg, 41%).

C₃₃ H₃₉ N₅ O₆ S [633.7]; [MH⁺ =634]

EXAMPLE 20

(RS)-N-[2-(Acetylmercapto)-5-phthalimidopentanoyl]-(S-methyl)-L-cysteinyl-L-phenylalanineN-methyl amide

From intermediate 36 (326 mg, 1 mmol) and intermediate 114 (350 mg, 1mmol), as a white solid (380 mg, 64%).

C₂₉ H₃₄ N₄ O₆ S₂ [598.7]; [MH⁺ =599]

EXAMPLE 21

(RS)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-(S)-propylglycinyl-L-phenylalanineN-methyl amide

From intermediate 113 (145 mg, 0.52 mmol) and intermediate 36 (168 mg,0.52 mmol), as a white solid (160 mg, 53%).

C₃₀ H₃₆ N₄ O₆ S [580.7]; [MH⁺ =581]

EXAMPLE 22

(RS)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-L-valinyl-N_(d)(1,1-dimethylethoxy)carbonyl-L-ornithine N-methyl amide

From intermedaite 112 (508 mg, 1.47 mmol) and intermediate 36 (491 mg,1.53 mmol), as a white solid (687 mg, 72%).

C₃₁ H₄₅ N₅ O₈ S [647.8]; [MH⁺ =648]

EXAMPLE 23

(RS)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-L-valinyl-L-ornithineN-methyl amide trifluoroacetate

A solution of example 22 (585 mg, 0.90 mmol) in dichloromethane (20 ml)containing trifluoroacetic acid (2 ml) was stirred at RT overnight. Thesolvent was removed in vacuo and any excess trifluoroacetic acid removedby azeotropic distillation with heptane (3×20 ml) to provide the titlecompound (596 mg, 99%) as a off white solid.

C₂₆ H₃₈ N₅ O₆ S [548.7]; [MH⁺ =549]

EXAMPLE 24

(RS)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-L-valinyl-N_(d)-(acetyl-L-ornithine N-methyl amide

Acetyl chloride (0.024 ml, 0.34 mmol) was added at 0° C. to a stirredsolution of example 23 (205 mg, 0.31 mmol) and N-methylmorpholine (0.134ml, 0.31 mmol) in anhydrous dichloromethane (20 ml). The mixture wasallowed to warm to RT and stirred for 1 h before diluting withdichloromethane (20 ml) and washing sequentially and 2N hydrochloride(20 ml), 8% sodium bicarbonate (20 ml), water (20 ml) and brine (20 ml),dried (MgSO₄) and evaporated in vacuo to a colourless foam. Purificationby flash column chromatography (eluting with 3-5%methanol-dichloromethane) provided the title compound (30 mg, 17%) as awhite solid.

C₂₈ H₄₁ N₅ O₇ S [590.7]; [MH⁺ =591]

EXAMPLE 25

(RS)-N-[2-(Acetylmercapto)-4-phthalimidobutanoyl]-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

A solution of potassium thiolacatate (98 mg) in methanol (2 ml) wasadded to a suspension of Intermediate 40 (0.5 g) in methanol (10 ml) andthe mixture was stirred at RT for 30 min, then at reflux for 6 h. Thesolvent was then removed under reduced pressure and the residue waspartitioned between water (50 ml) and dichloromethane (150 ml). Thelayers were separated and the organic layer was dried over sodiumsulfate, filtered, and evaporated to give the crude product.Purification by flash column chromatography (eluting with 50% ethylacetate-dichloromethane) gave the title compound as a colourless solid(270 mg).

TLC R_(f) 0.30 (50% EtOAc--CH₂ Cl₂)

Similarly prepared were:

EXAMPLE 26

(RS)-N-[2-(Acetylmercapto)-5-phthalimidopentanoyl]-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

From Intermediate 41 (0.5 g) as a near colourless solid (0.42 g).

TLC R_(f) 0.20 (50% EtOAc--CH₂ Cl₂)

EXAMPLE 27

(RS)-N-[2-(Acetylthio-6-phthalimidohexanoyl]-_(L) -leucyl-_(L)-phenylalamine N-methyl amide

From Intermediate 42 (0.50 g), as a plae yellow solid (0.39 g).

TLC R_(f) 0.31 (50% EtOAc--CH₂ Cl₂)

EXAMPLE 28

(RS)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-_(L) -leucyl-_(L)-[β-(4-thiazolyl)]alanine N-methyl amide

EDC (107 mg, 0.56 mmol) was aded to astirred solution of intermediate122 (222 mg, 0.51 mmol), N-hydroxybenzotriazole 76 mg, 0.56 mmol),triethylamine (75 ml, 0.53 mmol) and intermediate 100 (113 mg, 0.51mmol) in dry tetrahydrofuran (30 ml). The mixture was stirred at RTovernight then the solvent removed in vacuo and the residue partitionedbetween water (20 ml) and ethyl acetate (20 ml). The aqueous portion wasthen extracted with ethyl acetate (2×20 ml), the combined extractswashed with water (2×50 ml) and brine (50 ml), dried (MgSO₄) andevaporated in vacuo to a pale yellow oil.

Purification by column chromatography eluting withdichloromethane/methanol (98.2) provided the title compound (170 mg,55%) as a white solid.

C₂₈ H₃₅ N₅ O₆ S₂ [601.7]; [MH⁺ =602]

Similarly prepared were:

EXAMPLE 29

(RS)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-_(L) -leucyl-_(L)-[β-(2-pyridyl)]alanine N-methyl amide

From intermediate 122 and intermediate 99, as a white solid (277 mg,67%).

C₃₀ H₃₇ N₅ O₆ S [595.7]; [MH⁺ =596]

EXAMPLE 30

(RS)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-_(L) -leucyl-_(L)-[β-(3-pyridyl)]alanine N-methyl amide

From intermediate 122 and intermediate 97, as a white solid (50 mg,12%).

C₃₀ H₃₇ N₅ O₆ S [595.7]; [MH⁺ =596]

EXAMPLE 31

(RS)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-_(L) -leucyl-_(L)-[β-(4-pyridyl)]alanine N-methyl amide

From intermediate 122 and intermediate 98, as a white solid (310 mg,77%).

C₃₀ H₃₇ N₅ O₆ S [595.7]; [MH⁺ =596]

EXAMPLE 32

(RS)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-_(L)-leucyl-5-methyl-_(L) -glutamic acid N-methyl amide

From intermediate 122 and intermediate 103, as a white solid (201 mg,49%).

C₂₈ H₃₈ N₄ O₈ S [590.3]; [MH⁺ 591]

EXAMPLE 33

(RS)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-_(L)-leucyl-(S)-(2-methoxyphenyl)alanine N-methyl amide

From intermediate 122 and intermediate 102, as a white solid (150 mg,37%).

C₃₂ H₄₀ H₄ O₇ S [624.8]; [MH⁺ =625]

EXAMPLE 34

(RS)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-_(L)-leucyl-(R)-penicillamine N-methyl amide

From intermediate 122 and intermediate 96, as a white solid (230 mg,35%).

C₂₇ H₃₈ N₄ O₆ S₂ [578.8]; [MH⁺ =579]

EXAMPLE 35

(RS)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-_(L)-leucyl-(R)-(S-methyl)-penicillamine N-methyl amide

From intermediate 122 and intermediate 101, as a white solid (400 mg,35%).

C₂₈ H₄₀ N₄ O₆ S₂ [592.8]; [MH⁺ =593]

EXAMPLE 36

(RS)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-_(L)-leucyl-(R)-(2,2-dimethyl-2-methanesulphonyl)alanine N-methyl amide

From intermediate 122 and intermediate 95, as a white solid (380 mg,66%).

C₂₈ H₄₀ N₄ O₈ S₂ [624.8]; [MH⁺ =625]

EXAMPLE 37

(RS)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-_(L)-leucyl-(R)-(2,2-dimethyl-2-methanesulphinyl)alanine N-methyl amide

From intermediate 122 and intermediate 94, as a white solid (360 mg,37%).

C₂₈ H₄₀ N₄ O₇ S₂ [608.8]; [MH⁺ =609]

EXAMPLE 38

(RS)-N-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-_(L)-leucyl-(S)-tert-leucine N-methyl amide

From intermediate 122 and tert-leucine N-methyl amide hydrochloride, asa white solid (120 mg, 25%).

C₂₈ H₄₀ N₄ O₆ S [560.7]; [MH⁺ =561]

EXAMPLE 39

(RS)-N-[2-Mercapto-5-(acetyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

0.4M Sodium hydroxide (0.82 ml) was added to a stirred solution ofintermediate 57 (203 mg, 0.328 mmol) and 2-mercaptoethanol (0.23 ml,3.38 mmol) in a methanol at 0° C. After 15 min acetic acid (0.5 ml) wasadded and the solvents evaporated in vacuo to provide a yellow oil.Ether (20 ml) was added and the resulting precipitate removed byfiltration to provide the crude thiol. Purification by flash columnchromatography (eluting with 5% methanol in dichloromethane) providedthe title compound (86 mg, 56%) as a white solid.

C₂₃ H₃₆ H₄ O₄ S [464.6]; [MH⁺ =465]

Similarly prepared were:

EXAMPLE 40

(RS)-N-[2-Mercapto-5-(benzoyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 58 (400 mg, 0.59 mmol), as a white solid (175 mg,59%).

C₂₈ H₃₈ N₄ O₄ S [526.7]; [MH⁺ =527]

EXAMPLE 41

(RS)-N-[2-Mercapto-5-(succinimido)pentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 59 (600 mg, 0.91 mmol), as a white solid (147 mg,32%).

C₂₅ H₃₆ N₄ O₅ S [504.7]; [MH⁺ =505]

EXAMPLE 42

(RS)-N-[2-Mercapto-5-(methanesulphonyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 60 (600 mg, 1.0 mmol), as a white solid (262 mg, 57%).

C₂₂ H₃₆ N₄ O₅ S₂ [500.7]; [MH⁺ =501]

EXAMPLE 43

(RS)-N-[2-Mercapto-5-(benzoylphonyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 61 (500 mg, 0.7 mmol), as a white solid (221 mg, 56%).

C₂₇ H₃₆ N₄ O₅ S₂ [562.8]; [MH⁺ =563]

EXAMPLE 44

(RS)-N-[2-Mercapto-5-(4-pyridylcarbonyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 64 (330 mg, 0.48 mmol), as a white solid (115 mg,22%).

C₂₇ H₃₇ N₅ O₄ S [527.7]; [MH⁺ =528]

EXAMPLE 45

(RS)-N-[2-Mercapto-5-(3-pyridylcarbonyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 65 (200 mg, 0.29 mmol), as a white solid (34 mg, 22%).

C₂₇ H₃₇ N₅ O₄ S [527.7]; [MH⁺ =528]

EXAMPLE 46

(RS)-N-[2-Mercapto-5-(2-pyridylcarbonyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 66 (150 mg, 0.22 mmol), as a white solid (60 mg, 52%).

C₂₇ H₃₇ N₅ O₄ S [527.7]; [MH⁺ =528]

EXAMPLE 47

(RS)-N-[2-Mercapto-5-(2-pyrazinylcarbonyl)aminopentanoyl]-L-leucyl-L-phenylalanineN-methyl amide

From intermediate 67 (100 mg, 0.15 mmol), as a white solid (30 mg, 38%).

C₂₆ H₃₆ N₆ O₄ S [528.7]; [MH⁺ =529]

EXAMPLE 48

(RS)-N-[2-Mercapto-5-phthalimido]pentanoyl]-_(L) -leucyl-_(L)-[β-(4-thiazolyl)]alanine N-methyl amide

Concentrated ammonium hydroxide (0.5 ml) was added to a solution ofexample 28 (110 mg, 0.18 mmol) in methanol (10 ml) at 0° C. and themixture was stirred at that temperataure for 3 h. The mixture wasdiluted with water (10 ml), acidified with 2N aqueous hydrochloric acidand extracted with dichloromethane (3×20 ml). The combined extracts weredried (Na₂ SO₄) filtered and evaporated to give the crude product.Purification by flash column chromatography (eluting with 2%methanoldichloromethane) gave the title compound as a colourless solid(71 mg, 71%).

C₂₆ H₃₃ N₅ O₅ S₂ [559.7]; [MH⁺ =560]

Similarly prepared were:

EXAMPLE 49

(RS)-N-[2-Mercapto-5-phthalimido]pentanoyl-_(L) -leucyl-_(L)-[β-(2-pyridyl)]alanine N-methyl amide

From example 29, as a white solid (75 mg, 80%).

C₂₈ H₃₅ N₅ O₅ S [553.7]; [MH⁺ =554]

EXAMPLE 50

(RS)-N-[2-Mercapto-5-phthalimido]pentanoyl-_(L) -leucyl-5-methyl-_(L)-glutamic acid N-methyl amide

From example 32, as a white solid (68 mg, 78%).

C₂₆ H₃₆ N₄ O₇ S [548.6]; [MH⁺ =549]

EXAMPLE 51

(RS)-N-[2-Mercapto-5-phthalimido]pentanoyl-_(L)-leucyl-(R)-(S-methyl)-penicillamine N-methyl amide

From example 35, as a white solid (160 mg, 70%).

C₂₆ H₃₈ N₄ O₅ S₂ [550.8]; [MH⁺ =551]

EXAMPLE 52

(S)-N-[2-Mercapto-5-phthalimido]pentanoyl-_(L) -leucyl-(S)-tert-leucineN-methyl amide

From example 16, as a white solid (8.12 g, 80%).

C₂₆ H₃₈ N₄ O₅ S [518.7]; [MH⁺ =519]

EXAMPLE 53

(S)-N-[2-Mercapto-5-phthalimido]pentanoyl-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

From example 17, as a white solid (2.7 g, 67%).

C₂₉ H₃₆ N₄ O₅ S [552.7]; [MH⁺ =553]

EXAMPLE 54

(RS)-N-[2,3-Dimercaptopropanoyl]-_(L) -leucyl-_(L) -phenylalanineN-methyl amide

From example 1, as a colourless solid (76 mg).

C₁₉ H₂₉ N₃ O₃ S₂ [411.5]; [MH⁺ =412]

EXAMPLE 55

(RS)-N-[2-Mercapto-3-methoxycarbonylpropanoyl]-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

From example 5 (0.16 g), as a colourless solid (0.14 g).

C₂₁ H₃₁ N₃ O₅ S₂ [437.5]; [MH⁺ =438]

EXAMPLE 56

(RS)-N-[2-Mercapto-4-methoxycarbontylbutanoyl]-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

From example 6 (0.18 g), as a colourless solid (0.16 g).

C₂₂ H₃₃ N₃ O₅ S [451.5]; [MH⁺ =452]

EXAMPLE 57

(RS)-N-[2-Mercapto-5-methoxycarbontylpentanoyl]-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

From example 7 (0.32 g),as a colourless solid (0.15 g).

TLC R_(f) 0.29 (5% MeOH--CH₂ Cl₂)

EXAMPLE 58

(RS)-N-[2-Mercapto-6-methoxycarbontylhexanoyl]-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

From example 8 (0.31 g), as a colourless solid (0.22 g).

TLC R_(f) 0.30 (5% MeOH--CH₂ Cl₂)

EXAMPLE 59

(RS)-N-[2-Mercapto-4-phthalimidobutanoyl]-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

From example 25 (0.20 g), as a pale yellow solid (0.12 g).

C₂₈ H₃₄ N₄ O₅ S [538.6]; [MH₂ ⁺ =540]

EXAMPLE 60

(RS)-N-[2-Mercapto-5-phthalimidopentanoyl]-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

From example 26 (0.2 g), a a colourless solid (0.18 g).

TLC R_(f) 0.41 (5% MeOH--CH₂ Cl₂)

EXAMPLE 61

(RS)-N-[2-Mercapto-6-phthalimidopentanoyl]-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

From example 27 (0.15 g), as a pale yellow solid (0.12 g).

TLC R_(f) 0.25 (10% MeOH--CH₂ Cl₂)

EXAMPLE 62

(RS)-N-[2-Mercapto-5-phthalimidopentanoyl]-_(L) -leucyl-_(L)-tryptophanN-methyl amide

From example 9 (250 mg), as a pale yellow foam (222 mg, 96%).

C₃₁ H₃₇ N₅ O₅ S [591.7]; [MH⁺ =592]

EXAMPLE 63

(RS)-N-[2-Mercapto-6-methoxycarbonylhexanoyl]-_(L) -leucyl-_(L)-trypotophan N-methyl amide

From example 10 (330 mg), as a colourless foam (300 mg, 98%).

C₂₆ H₃₈ N₄ O₅ S [518.7]; [MH⁺ =519]

EXAMPLE 64

(S)-N-[2-Mercapto-5-phthalimidopentanoyl]-_(L) -(S-methyl)cysteinyl-_(L)-tryptophan N-methyl amide

Was prepared by the procedure described previously for examples 28 and48, from intermediate 119 and intermediate 124.

C₂₉ H₃₂ N₅ O₅ S₂ [595.7]; [MH⁺ =596]

EXAMPLE 65

(RS)-N-[2-Mercapto-3-phthalimidopentanoyl]-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

Was prepared by the procedure described previously for example 39 viaintermediate 43, from intermediate 125 and ^(L) -leucyl-_(L)-phenylalanine N-methyl amide.

C₂₇ H₃₂ N₄ O₅ S [524.6]; [MH⁺ =525]

EXAMPLE 66

(RS)-N-[2-Mercapto-4-succinimidobutanoyl]-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

Was prepared by the procedure described previously for examples 28 and48, from intermediate 123 and _(L) -leucyl-_(L) -phenylalanine N-methlyamide.

C₂₄ H₃₄ N₄ O₅ S [490.6]; [MH⁺ =491]

EXAMPLE 67

(RS)-N-[2-Mercapto-4-succinimidobutanoyl]-(S)-propylglycinyl-_(L)-phenylalanine N-methyl amide

Was prepared by the procedure described previously for examples 28 and48, from intermediate 123 and intermediate 113.

C₂₃ H₃₁ N₄ O₅ S [475.6]; [MH⁺ =476]

EXAMPLE 68

(RS)-N-[2-Mercapto-4-succinimidobutanoyl]-_(L) -(S-methyl)cysteinyl-_(L)-tert-leucine N-methyl amide

Was prepared by the procedure described previously for examples 28 and48, from intermediate 123 and intermediate 114.

C₁₉ H₃₂ N₄ O₅ S₂ [460.6]; [MH⁺ =461]

EXAMPLE 69

(RS)-N-[2-Mercapto-4-succinimidobutanoyl]-_(L) -(S-methyl)cysteinyl-_(L)-tryptophan N-methyl amide

Was prepared by the procedure described previously for examples 28 and48, from intermediate 123 and intermediate 124.

C₂₄ H₃₁ N₅ O₅ S₂ [533.7]; [MH⁺ =534]

EXAMPLE 70

(RS)-N-[2-Mercapto-4-succinimidobutanoyl]-_(L) -(S-methyl)cysteinyl-_(L)-phenylalanine N-methyl amide

Was prepared by the procedure described previously for examples 28 and48, from intermediate 123 and intermediate 111.

C₂₃ H₃₀ N₄ O₅ S₂ [506.6]; [MH⁺ =507]

EXAMPLE 71

(RS)-N-[2-Mercapto-4-succinimidobutanoyl]-_(L) -(O-methyl)serinyl-_(L)-tert-leucine N-methyl amide

Was prepared by the procedure described previously for examples 28 and48, from intermediate 123 and intermediate 115.

C₁₉ H₃₂ N₄ O₅ S [444.5]; [MH⁺ =445]

EXAMPLE 72

(RS)-N-[2-Mercapto-4-succinimidobutanoyl]-_(L) -leucyl-_(L)-tert-leucine N-methyl amide

Was prepared by the procedure described previously for examples 28 and48, from intermediate 123 and intermediate 116.

C₂₁ H₃₆ N₄ O₅ S [456.6]; [MH⁺ =457]

EXAMPLE 73

(RS)-N-[2-Mercapto-6-carboxyhexanoyl]-_(L) -leucyl-_(L) -tryptophanN-methyl amide

Was prepared by hydrolysis of example 10.

C₂₅ H₃₆ N₄ O₅ S [504.7]; [MH⁺ =505]

EXAMPLE 74

(R)-N-[2-Acetylmercapto-6-methoxycarbonylhexanoyl]-_(L) -leucyl-_(L)-trypophan N-methyl amide

Was prepared by separation of the 1:1 mixture of diastereoisomerspresent in example 10 by flash column chromatography.

C₂₆ H₃₈ N₄ O₅ S [518.7]; [MH⁺ =519]

EXAMPLE 75

(S)-N-[2-Acetylmercapto-6-methoxycarbonylhexanoyl]-_(L) -leucyl-_(L)-trypophan N-methyl amide

Was prepared by separation of the 1:1 mixture of diastereoisomerspresent in example 10 by flash column chromatography.

C₂₆ H₃₈ N₄ O₅ S [518.7]; [MH⁺ =519]

EXAMPLE 76

(R)-N-[2-Acetylmercapto-5-phthalimidopentanoyl]-_(L) -leucyl-_(L)-trypophan N-methyl amide

Was prepared by separation of the 1:1 mixture of diastereoisomerspresent in example 9 by flash column chromatography.

C₃₁ H₃₇ N₅ O₅ S₂ [591.7]; [MH⁺ =592]

EXAMPLE 77

(R)-N-[2-Acetylmercapto-5-methoxycarbonylhexanoyl]-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

Was prepared by separation of the 1:1 mixture of diastereoisomerspresent in example 7 by flash column chromatography.

C₂₅ H₃₆ N₄ O₅ S [504.7]; [MH⁺ =505]

EXAMPLE 78

(R)-N-[2-Mercapto-6-(methylamino)carbonylhexanoyl]-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

Was prepared by hydrolysis of example 8.

C₂₄ H₃₈ N₄ O₄ S [478.6]; [MH⁺ =479]

EXAMPLE 79

(RS)-N-[2-Mercapto-6-(amino)carbonylhexanoyl]-_(L) -leucyl-_(L)-phenylalanine N-methyl amide

Was prepared by hydrolysis of example 8.

C₂₃ H₃₆ N₄ O₄ S [464.6]; [MH⁺ =465]

EXAMPLE A

Collagenase inhibition activity

The potency of compounds of general formula (I) to act as inhibitors ofcollagenase was determined by the procedure of Cawston and Barrett,(Anal. Biochem., 99:340-345, 1979) whereby a 1 mM solution of theinhibitior being tested or dilutions thereof was incubated at 37° C. for16 hours with collagen and collagenase (buffered with 50 mM Tris, pH 7.6containing 5 mM CaCl₂, 0.05% Brij 35, 60 mM NaCl and 0.02% NaN₃). Thecollagen was acetylated ³ H or ¹⁴ C-collagen prepared by the method ofCawston and Murphy (Methods in Enzymology, 80:711, 1981). The choice ofradiolabel did not alter the ability of collagenase to degrade thecollagen substrate. The samples were centrifuged to sediment undigestedcollagen and an aliquot of the radioactive supernatant removed for assayon a scintillation counter as a measure of hydrolysis. The collagenaseactivity in the presence of 1 mM inhibitor, or a dilution thereof, wascompared to activity in a control devoid of inhibitior and the resultsreported as that inhibitor concentration effecting 50% inhibition of thecollagnease (IC₅₀).

EXAMPLE B

Stromelysin inhibition activity

The potency of compounds of general formula (I) to act as inhibitors ofstromelysin was determined using the prodecure of Nagase et al (Methodsin Enzymology Vol 254, 1994), whereby a 0.1 mM solution of the inhibitorbeing tested or dilutions thereof was incubated at 37° C. for 16 hourswith stromelysin and ³ H transferrin (buffered with 50 mM Tris, pH 7.6containing 10 mM CaCl₂, 150M NaCl, 0.05% Brij, 35, and 0.02% NaN₃). Thetansferin was carboxymethylated with ³ H iodoacetic acid. Thestromelysin activity in the presence of 1 mM, or a dilution thereof, wascompared to activity in a control devoid of inhibitor and the resultsreported as that inhibitor concentration effecting 50% inhibition of thestromelysin (IC₅₀)

EXAMPLE C

Gelatinase inhibition activity

The potency of the compounds of general formula (I) to act as inhibitorsof gelatinase was determined using the procedure of Harris & Krane(Biochem Biophys. Acta, 258:566-576, 1972), whereby a 1 mM solution ofthe inhibitior being tested or dilutions thereof was incubated at 37° C.for 16 hours with gelatinase and heat denatured ³ H or ¹⁴ C-acetylatedcollagen (buffered with 50 mM Tris, pH 7.6 containing 5 mM CaCl₂, 0.05%Brij 35 and 0.02% NaN₃). The ³ H or ¹⁴ C gelatin was prepared bydenaturing ³ H or ¹⁴ C-collagen produced according to the method ofCawston and Murphy (Methods in Enzymology, 80:711, 1981) by incubationat 60° C. for 30 minutes. Undigested gelatin was precipitated byaddition of trichloroacetic acid and centrifugaion. The gelatinaseactivity in the presence of 1 mM, or dilution thereof, was compared tothe activity in a control devoid of inhibitior and results reported asthat inhibitorconcentraion effecting 50% inhibition of the gelatinase(IC₅₀).

EXAMPLE D

Inhibition of TNFα production

The potency of the compounds of general formula (I) to act as inhibitorsof the production of TNFa was determined using the floowing procedure. A1 mM solution of the inhibitor being tested or dilutions thereof wasincubated at 37° C. in an atmosphere of 5% CO₂ with THP-1 cells (humanmonocytes) suspended in RPM1 1640 medium and 20 μM β-mercaptoethanol ata cell densiy of 1×10⁶ /ml and stimulated with 5 μg/ml finalconcentraion of LPS. After 18 hours the supernatant is assayed for thelevels of TNFα using aconmmercially available ELISA kit (R & D Systems).

The activity in the presence of 0.1 mM inhibitior or dilutions thereofwas compared to activity in a control devoid of inhibitior and resultsreported as that inhibitor concentraion effecting 50% inhibition of theproduction of TNFa.

EXAMPLE E

Adjuvant arthritic rat model

Compounds of general formula (I) were evaluated in an adjuvant arthritismodel in the rat based on the methods employed by B. B. Newbould (1963),Br. J. Pharmacol, 21, 127-136 and C. M. Pearson and F. D. Wood (1959),Arthritis Rheum, 2, 440-459. Briefly male Wistar rats (180-200 g) wereinjected at the base of the tail with Freund's adjuvant. Twelve dayslater the responding animals were randomised into experimental groups.Compounds of general formula (I) were dosed either orally as asuspension in 1% methyl cellulose or intraperitoneally in 0.2%carboxymethylcelluslose from day 12 to the end of the experiment on day22. Hind paw volumes were measured every two days from day 12 onwardsand X-rays were taken of the hind feet on completion of the experiment.Results were expressed as the percent increase of foot volume over day12 values.

EXAMPLE F

Mouse ovarian carcinoma xenograft model

Compounds of general formula (I) were evaluated in an ovarian carcinomaxenograft model of cancer, based on that described by B. Davies et al(1993), Cancer Reasearch, 53, 2087-2091 This model, in brief, consistsof inoculating female nu/nu mice with 1×10⁹ OVCAR3-icr cells into theperitoneal cavity. Compounds of general formula (I) are administered bytheoral route as a suspension in 1% methyl cellulose orintraperitoneally as a suspension in phosphate buffered saline in 0.01%Tween-20. At the conculusion of the experiment (4-5 weeks) the number ofperitoneal cells are counted and any solid tumour deposits weighted. Insome experiments tumour development is monitored by measurement oftumour specific antigens.

EXAMPLE G

Rat mammary carcinoma model

Compounds of general formula (I) were evaluated in a HOSP.1 rat mammarycarcinoma model of cancer (S. Eccles et al (1995), Cancer Research, inpress). This model consists of the intravenous inoculation of femaleCBH/cbi rats with 2×10⁴ tumour cells into the jugular vein. Compounds ofgeneral formula (I) are administered by the oral route as a suspensionin 1% methyl cellulose or intraperitoneally as a suspension in phosphatebuffered saline in 0.01% Tween-20. At the conclusion of the experiment(4-5 weeks) the animals were killed, the lungs were removed andindividual tumours counted after 20 hours fixation in Methacarn.

We claim:
 1. A compound of general formula (I): ##STR12## wherein: R¹ isC₁₋₆ alkyl, C₂₋₆ alkenyl, --C₁₋₆ alkyl-aryl, aryl, --C₁₋₆alkyl-heteroaryl, heteroaryl or --C₁₋₆ alkyl-AR⁹ where A represents O,NR⁹ or S(O)_(m) where m=0.2, and R⁹ is H, C₁₋₄ alkyl, aryl heteroryl,--C₁₋₄ alkyl-aryl or --C₁₋₄ alkyl-heteroaryl; if A=NR⁹ the groups R⁹ maybe the same or different;R² is H or C₁₋₆ alkyl; R³ is [Alk]_(n) R⁶ whereAlk is C₁₋₆ alkyl or C₂₋₆ alkenyl or n is zero or 1; X is NR⁴ R⁵ whereeither R⁴ is hydrogen or C₁₋₆ alkyl optionally substituted by amino(NH₂), aryl, arylamino, protected amino, di(C₁₋₆ alkyl)amino, momo(C₁₋₆alkyl)amino, CO₂ H, protected carboxyl, carbomoyl, mono(C₁₋₆alkyl)carbamoyl or di(C₁₋₆ alkyl)carbamoyl, and R⁵ is hydrogen or C₁₋₆alkyl; or NR⁴ R⁵ forms a ring such as pyrrolidino, piperidino ormorpholino; R⁷ is hydrogen or R¹⁰ CO where R¹⁰ is C₁₋₄ alkyl, --C₁₋₄alkyl-aryl, --C₁₋₄ alkyl-heteroaryl, cyclo (C₃₋₆)alkyl, --C₁₋₄alkyl-cyclo(C₃₋₆)alkyl, C₂₋₆ alkenyl, --C₂₋₆ alkenyl-aryl, aryl, orheteroaryl; R⁸ is aryl substituted with R¹¹, heteroaryl substituted withR¹¹, C₁₋₄ alkyl-R¹¹, --C₁₋₄ alkyl-aryl substituted with R¹¹, --C₁₋₄alkyl-heteroaryl substituted with R¹¹, cyclo(C₃₋₆)alkyl substituted withR¹¹, cyclo(C₃₋₆)alkenyl substituted with R¹¹, --C₁₋₄alkyl-cyclo(C₃₋₆)alkyl substituted with R¹¹, or any of the three groups##STR13## where p is 1 or 2 and B and C are independently selected fromO, S, C(R⁹)₂ and NR⁹ ; R⁶ is AR⁹, cyclo(C₃₋₆)alkyl, cyclo(C₃₋₆)alkenyl,C₁₋₆ alkyl, --C₁₋₆ alkoxy-aryl, benzyloxyaryl, aryl, heteroaryl, --C₁₋₃alkyl-heteroaryl, --C₁₋₃ alkyl-aryl, --C₁₋₆ alkyl-COOR⁹, --C₁₋₆alkyl-NHR, CONHR, NHCO₂ R, NHSO₂ R or NHCOR, R being defined as for R¹⁰; R¹¹ is COR¹³, succinimido or phthalimido; R¹² is H or COR⁹, CO₂ R⁹(where R⁹ is not H), CONHR⁹ or SO₂ R⁹ (where R⁹ is not H); and R¹³ isOH, OC₁₋₄ alkyl, O--C₁₋₄ alkyl-aryl, N(R⁹)₂ (in which the R⁹ s are thesame or different), C₁₋₄ alkyl, aryl, heteroaryl, --C₁₋₄ alkyl-aryl or--C₁₋₄ alkyl-heteroaryl; the compound being in the form of a non-salt,salt, solvate, or hydrate.
 2. A compound of claim 1, wherein R¹ is C₁₋₆alkyl or C₁₋₆ alkyl-AR⁹, where A is S(O)_(m), NR⁹, or O; and m=0, 1, or2; and R⁹ is H, C₁₋₄ alkyl or aryl.
 3. A compound of claim 1, wherein R³is [Alk]_(m) R⁶ where n=0 or 1, Alk is C₁₋₆ alkyl, and R⁶ is C₁₋₆ alkyl,--C₁₋₃ alkyl-aryl, --C₁₋₃ alkyl-heteroaryl, or AR⁹.
 4. A compound ofclaim 1, wherein R⁴ is H.
 5. A compound of claim 1, wherein X ispyrrolidino, piperidino, or morpholino.
 6. A compound of claim 1,wherein R⁷ is H or (C₁₋₄)carbonyl.
 7. A compound of claim 1, wherein R⁸is C₁₋₄ alkyl-R¹¹ or cyclo (C₃₋₆)alkyl-R¹¹, R¹² is COR⁹, CO₂ R⁹(provided R⁹ is not H) or SO₂ R⁹ (provided R⁹ is not H), and R¹³ is OH,OC₁₋₄ alkyl or N(R⁹)₂.
 8. A compound of claim 1, wherein R⁵ is H or C₁₋₆alkyl.
 9. A compound of claim 8, wherein:R¹ is alkyl, alkenyl,alkylaryl, aryl or alkyl-AR⁹ and R⁹ is alkyl, aryl or heteroaryl; R⁷ isH or R¹⁰ CO where R¹⁰ is alkyl, alkylaryl, cycloalkyl, cycloalkylakyl,alkenyl or alkenylaryl; R⁸ is aryl substituted with R¹¹, heteroarylsubstituted with R¹¹, --C₁₋₄ alkylaryl substituted with R¹¹,cyclo(C₃₋₆)alkyl substituted with R¹¹, cyclo(C₃₋₆)alkenyl substitutedwith R¹¹ or --C₁₋₄ alkylcyclo(C₃₋₆)alkyl substituted with R¹¹, alkyl-R¹¹or any of the said three groups; R⁶ is cycloalkyl, cycloalkenyl, alkyl,benzyl, alkoxybenzyl or 3- indolylmethyl; and R¹³ is OH, Oalkyl,Oalkylaryl, N(R⁹)₂ alkyl, aryl or alkylaryl.
 10. A compound of claim 1,selected from the group consistingofN-[2,3-bis-Acetylmercaptopropanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Acetylmercapto-3-methoxycarbonylpropanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Acetylmercapto-4-methoxycarbonylpropanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Acetylmercapto-5-methoxycarbonylpropanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Acetylmercapto-6-methoxycarbonylhexanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Acetylmercapto-4-phthalimidobutanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Acetylmercapto-5-phthalimidopentanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Acetylmercapto-6-phthalimidohexanoyl]-L-leucyl-L-phenylalanineN-methylamide N-[2,3-bis-Mercaptopropanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Mercapto-3-methoxycarbonylpropanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Mercapto-4-methoxycarbonylbutanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Mercapto-5-methoxycarbonylpentanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Mercapto-6-methoxycarbonylhexanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Mercapto-4-phthalimidobutanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Mercapto-5-phthalimidopentanoyl]-L-leucyl-L-phenylalanineN-methylamide andN-[2-Mercapto-6-phthalimidohexanoyl]-L-leucyl-L-phenylalanineN-methylamide.
 11. A compound of claim 1, selected from the groupconsistingofN-[2-Acetylmercapto-5-methoxycarbonylpentanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Acetylmercapto-6-methoxycarbonylhexanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Acetylmercapto-6-methoxycarbonylhexanoyl]-L-valinyl-L-phenylalanineN-methylamideN-[2-Acetylmercapto-6-methoxycarbonylhexanoyl]-L-leucyl-L-tryptophanN-methylamideN-[2-Acetylmercapto-5-phthalimidopentanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Acetylmercapto-5-phthalimidopentanoyl]-L-valinyl-L-phenylalanineN-methylamideN-[2-Acetylmercapto-5-phthalimidopentanoyl]-L-leucyl-L-tryptophanN-methylamideN-[2-Acetylmercapto-5-phthalimidopentanoyl]-L-leucyl-L-[β-(4-thiazolyl)]alanineN-methylamideN-[2-Acetylmercapto-5-phthalimidopentanoyl]-L-leucyl-L-[β-(2-pyridyl)]alanineN-methylamideN-[2-Acetylmercapto-5-phthalimidopentanoyl]-L-leucyl-5-methyl-L-glutamicacid N-methylamideN-[2-Acetylmercapto-6-phthalimidohexanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Acetylmercapto-2-(3-phthalimido)phenylacetyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Mercapto-5-methoxycarbonylpentanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Mercapto-6-methoxycarbonylhexanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Mercapto-6-methoxycarbonylhexanoyl]-L-leucyl-L-trypotophanN-methylamideN-[2-Mercapto-5-phthalimidopentanoyl]-L-leucyl-L-phenylalanineN-methylamideN-[2-Mercapto-6-phthalimidopentanoyl]-L-leucyl-L-tryptophanN-methylamideN-[2-Mercapto-5-phthalimidopentanoyl]-L-leucyl-L-[β-(4-thiazolyl)]alanineN-methylamideN-[2-Mercapto-5-phthalimidopentanoyl]-L-leucyl-L-[β-(2-pyridyl)]alanineN-methalamide andN-[2-Mercapto-5-phthalimidopentanoyl]-L-leucyl-5-methyl-L-glutamic acidN-methylamide andN-[2-Mercapto-6-phthalimidohexanoyl]-L-leucyl-L-phenylalanineN-methylamide.
 12. A compound of claim 1, wherein R¹ is --CH₂ SCH₃. 13.A compound of claim 12, selected from the group consistingofN-[2-(Acetylmercapto)-5-phthalimidopentanoyl]-(S-methyl)-L-cyseinyl-L-phenylalanineN-methyl amideN-[2-Mercapto-5-phthalimidopentanoyl]-L-(S-methyl)cysteinyl-L-tryptophanN-methyl amideN-[2-Mercapto-4-succinimidobutanoyl]-L-(S-methyl)cysteinyl-L-tert-leucineN-methyl amideN-[2-Mercapto-4-succinimidobutanoyl]-L-(S-methyl)cysteinyl-L-tryptophanN-methyl amide andN-[2-Mercapto-4-succinimidobutanoyl]-L-(S-methyl)cysteinyl-phenylalanineN-methyl amide.
 14. A compound of claim 1, wherein R³ is tert-butyl or--C(CH₃)₂ S(O)₀₋₂ CH₃.
 15. A compound of claim 14, selected from thegroup consisingofN-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-L-leucyl-(S)-tert-leucineN-methyl amideN-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-L-leucyl-(R)-penicillamineN-methyl amideN-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-L-leucyl-(R)-(S-methyl)-penicillamineN-methyl amideN-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-L-leucyl-(R)-(2,2-dimethyl-2-methanesulphonyl)alanineN-methyl amideN-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-L-leucyl-(R)-(2,2-dimethyl-2-methanesulphinyl)alanineN-methyl amideN-[2-(Acetylmercapto)-5-phthalimido]pentanoyl-L-leucyl-(R)-(S)-tert-leucineN-methyl amideN-[2-Mercapto-5-phthalimido]pentanoyl-L-leucyl-(R)-(S-methyl)-penicillamineN-methyl amideN-[2-Mercapto-5-phthalimido]pentanoyl-L-leucyl-(S)-tert-leucine N-methylamide and N-[2-Mercapto-4-succinimidobutanoyl]-L-leucyl-L-tert-leucineN-methyl amide.
 16. A compound of claim 1, in the form of a singleenantimoer or diastereomer, or a mixture of such isomers.
 17. Apharmaceutiacl composition for use in therapy, comprising a compound ofclaim 1, and a pharmaceutically-acceptable diluent or carrier.
 18. Amethod for the treatment of a condition associated with matrixmetalloproteinases or that is mediated by TNFα, said method comprisingadministering an effective amount of the compound of claim 1 to a personor animal in need of such treatment.
 19. The method according to claim18, wherein the condition is selected from the group consisting ofinflammation and inflammatory diseases, tissue degeneration, periodontaldisease, ophthalmological disease, dermatological disorders, fever,cardiovascular effects, haemorrhage, coagulation and acute phaseresponse, cachexia and anorexia, acute infection, HIV infection, shockstates, graft versus host reactions,autoimmune disease, reperfusioninjury, meningitis and migraine.
 20. The method according to claim 19,wherein the condition is selected from the group consisting of tumourgrowth, angiogenesis, tomour invasion and spread, metastases, malignantascites and malignant pleural effusion.
 21. The method according toclaim 19, wherein the condition is selected from the group consisting ofrheumatoid arthritis, osteosrthritis, osteoporosis, asthma, multiplesclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis,stroke, vasculitis, Crohn's disease and ulcerative colitis.
 22. Themethod according to claim 19, wherein the condition is selected from thegroup consisting of corneal ulceration, retinopathy and surgical woundhealing.
 23. The method according to claim 19, wherein the condition isselected from the group consisting of psoriasis, atopic dermatitischronic ulcers and epidermolysis bullosa.
 24. The method according toclaim 19, wherein the condition is periodonititis or gingivitis.